EDTA Therapy for Vascular Disease

Chelation Natural Miracle For Protecting Your Heart

Chelation therapy has been conclusively shown to be up to 82 Percent Effective at dissolving the plaque that blocks arteries! In the ebook Chelation Natural Miracle For Protecting Your Heart you'll discover: The frustrating reason many doctors are ignoring Edta chelationor even openly rejecting it. The deadly heart surgeries even the American Heart Association admits are unnecessary. The hidden signs and symptoms of heart attacks and strokes? Are you in danger right now?. The average number of years stripped away by heart and vessel disease. Can you get them back?. The newest set of risk factors for heart disease (they'll likely surprise you!). Shady government practices that protect Big Pharma and keep Edta chelation out of the public eye. How the Roman Empire could have been savedif only they'd known about Edta chelation. Why Edta chelation is guaranteed to be safeeven in extremely high doses. (It puts aspirin to shame!). The shocking truth about plaque in young childrenand how to keep your little ones safer. Why dentists, artists and welders need Edta chelationand whether your workplace is dangerous too. The differences between IV and oral chelationand which kind of Edta is right for you. Other forms of chelationand how these little-known treatments can dramatically boost your health.

Chelation Natural Miracle For Protecting Your Heart Overview


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EDTA Treatment of the Membrane Fraction Carrying PQQ as Coenzyme

After a membrane suspension (10 mg of protein per mL) is mixed with 20 mmol L-1 EDTA for 30 min in an ice bath. The excess EDTA is removed from the membrane by ultracentrifugation two or three times at 68 000 x g for 60 min. The precipitate is resuspended in a buffer to wash EDTA out of the membrane fraction, followed by ultracentrifugation again under the same conditions. The resulting precipitate is resuspended with the same buffer. Under these conditions, as mentioned below, many PQQ-dependent dehydrogenases are resolved to apoen-zymes. However, some PQQ-dependent dehydrogenases are still active, although some decrease in enzyme activity is observed. If no loss of enzyme activity is found, the presence of covalently bound FAD as the coenzyme is the alternative possi-bility. To convert the apoenzyme to the holoenzyme, PQQ and divalent cations such as Ca2+ or Mg2+ are added to 5 imol L-1 and 5 mmol L-1, respectively, and the enzyme incubated, for example, for 30 min at 25 C, until...

Recipes O Edta

To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH. 10 mM Tris-Cl (pH 8.0) 0.1 M NaCl 1 mM EDTA (pH 8.0) 10 mM Tris-Cl (pH 8.0) 0.1 M NaCl 1 mM EDTA (pH 8.0) 5 (v v) Triton X-100 100 mM Tris-Cl (desired pH) 10 mM EDTA (pH 8.0) (10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg cm 2) on liquid cycle. Store the buffer at room temperature.

EDTATrisDNA solution

Dissolve carrier DNA (e.g., salmon sperm DNA) at a concentration of 50 Mg ml in 50 mM EDTA-Tris (pH 6.0) (i.e., a 1 10 dilution of the 0.5 M EDTA-Tris solution). The DNA used as carrier should be of fairly uniform size and not interfere with the hybridization of the radiolabeled nucleic acid to its target DNA. For most purposes, sonicated or fractionated salmon sperm DNA works well (sonicated salmon sperm DNA 600-5000 bp in length may be prepared as described in Chapter 6, Protocol 10).

Nuclear Transfer to Unfertilized Enucleated Eggs

Dissect a donor embryo (such as a late blastula) to isolate the pigmented layer of cells called an animal cap (see Fig. 3). Place the animal cap in the agarose-lined culture dish containing CaMg-free MBS. 1 mM ethylene diamine tetraacetic acid (EDTA) can be added to accelerate cell dissociation.

IncludedACGIH Region Regions RegionRegion

Various factors modify absorption from the GI tract and enhance or depress its barrier function. A decrease in gastrointestinal mobility generally favors increased absorption. Specific stomach contents and secretions may react with the contaminant and possibly change it to a form with different physicochemical properties (e.g., solubility), or they may absorb it, alter the available chemical, and change the translocation rates. The size of ingested particulates also affects absorption. Because the rate of dissolution is inversely proportional to particle size, large particles are absorbed to a lesser degree, especially if they are fairly insoluble in the first place. Certain chemicals, e.g., chelating agents such as EDTA, also cause a nonspecific increase in the absorption of many materials.

Introduction To Free Radicals And Antioxidants

In general, to be called antioxidant, a compound must be able to donate an electron and or hydrogen atom and prevent or delay oxidation of an oxidizable substrate. They can act in different ways by metal chelation (preventing free radical formation), scavenging free radicals, acting as a chain-breaking (stopping propagation of the free radicals), being part of the redox antioxidant network, and or regulating gene expression.

Uptake and Translocation of Nickel by Plants

Substrate was enhanced in the presence of copper 146 , while Cataldo et al. 160 found that Ni absorption by hydroponically grown soybean plants (Glycine max (L.) Merr. cv. Williams) was completely inhibited in the presence of Cu. Singh et al. 161 observed that an increasing supply of urea appeared to mitigate the toxicity of Ni to wheat (Triticum aestivum L.). The presence of organic acids or inorganic ligands in soil solution results in formation of Ni complexes, which may either inhibit or enhance root uptake, depending on the characteristics of the Ni complex formed. For example, Molas 162 reports that Ni(II)-EDTA was less enriched and less toxic than Ni(II)-citrate or Ni(II)-Glu to hydroponically grown cabbage plants. Generally, the ionic form of Ni2+ is taken up relatively easily by plants, but many chelated forms appear to be less available 160,163 . High-molecular-weight solutes are prevented from entering the apoplasm of root cells by the diameter of pores 143 . Plants can...

Diversities of Complement Components C4A and C4B in Human Populations

About 40 polymorphic protein variants of human C4 have been detected, based on gross differences in electric charge and serologic variations. The most widely used method for C4 phenotyping is immunofixation of EDTA-plasma proteins resolved by high-voltage agarose gel electrophoresis (Figure 7.1C). The most common C4A and C4B allotypes are C4A3 and C4B1, respectively. Other common allotypes for C4A include A2, A4, and A6, and for C4B, B2, B3, and B5. These allotypes exhibit different frequencies among different races or ethnic groups. For example, in the Ohio population, C4B2 has a frequency of 9.4 in Caucasians, but 33.6 in Asian Chinese (Yang et al., 2003 Yang, 2004).

Importance of Partitioning for Bioavailability and Mobility

Total Ni contents in the soil might depict the potential availability of Ni, but in most cases the total content is of little relevance in terms of Ni availability. The basic aim of a number of soil extraction schemes is to estimate the metal pool available to plants or other soil biota 170 . Single extractants such as dilute acids (HCl, NH4, HNO3) or solutions containing chelating agents (DTPA or EDTA) are traditionally used to estimate the bioavailable proportion of soil metal 7 . However, when applied to soils with a wide range of soil characteristics, they fail to predict metal bioavailability adequately 171 . In addition to the mineralogi-cal form of a metal, the key properties controlling its solubility and availability are soil solution pH, dissolved organic matter, and solid-phase metal oxide and organic matter content 172 .

Transfusion haemosiderosis

Iron overload occurs in patients with transfusion dependent anaemia, notably thalassaemia major, Diamond-Blackfan syndrome, aplastic anaemia and acquired refractory anaemia. In many of these conditions iron overload is aggravated by physiological mechanisms which promote 130 increased dietary absorption of iron in response to ineffective erythro-poiesis. Each unit of blood contains 250mg iron and average transfusion dependent adult receives 6-10g of iron year. Distribution of iron is similar to haemochromatosis with primarily liver parenchymal cell accumulation followed by pancreas, heart and other organs. Cardiac deposition occurs in patients who have received 100 units of blood (20g iron) without chelation, and is followed by damage to the liver, pancreas and endocrine glands.

Advanced Concepts

Sometimes, DNA preparations are intended for long-term storage. The presence of the chelating agent EDTA protects the DNA from damage by DNAses present in the environment. EDTA is a component of TE buffer (10 mM Tris, 1 mM EDTA) and other resuspension buffers. The EDTA will also inhibit enzyme activity when the DNA is used in various procedures such as restriction enzyme digestion or polymerase chain reaction (PCR). One must be careful not to dilute the DNA too far so that large volumes (e.g., more than 10 of a reaction volume) of the DNA-EDTA solution are required. When DNA yield is low, as is the case with some clinical samples, it is better to dissolve it in water. More of this can be used in subsequent procedures without adding excess amounts of EDTA. Because the entire sample will be used for analysis, protection on storage is not a concern.

Preparation and Quality Control of Nano Sized Dendrimer Based Paramagnetic MRI Contrast Agents

The details of preparation methods for each agent have been previously published in Kobayashi et al. (2003b, 2001a,b,d). In order to load maximum gadolinium ions on a single dendrimer molecule, minor modification of reaction condition was made in each dendrimer. Other researchers used similar but different synthetic methods and chelates, and reported variable conjugation ratio of the chelate molecules and gadolinium ions to dendrimer molecules (Bryant et al., 1999 Wiener et al., 1994). All of the dendrimers investigated for MRL ranged from generation-2 (G2 3 nm) to generation-10 (G10 15 nm) for polyami-doamine (PAMAM) and from generation-2 to generation-4 for polypropy-lenimine diaminobutane (DAB). The dendrimer substrate was obtained from commercial sources (Dendritech, Inc., Midland, MI or Aldrich Chemical Co., Milwaukee, WI). These compounds are highly soluble in aqueous solution and both possess a spherical surface topology composed of primary amino groups that increase...

Materials and Methods

For analysis of spliceosomes by gel filtration, large-scale splicing reactions (750 pd) were performed containing 0.5 mM ATP, 20 mM phosphocreatine, 2 mM MgCl2, 0.5 mMDTT, 1 ng ,1 capped, uniformly 32P-labeled pre-mRNA substrate (transcribed as above), 40 HeLa nuclear extract in buffer E (20 mM Tris pH 7.9, 100 mM KC1, 0.2 mM EDTA, 0.5 mM DTT, 20 glycerol), and additional buffer E to obtain a final concentration of 60 mM KC1. E complex assembly reactions were performed under identical conditions, except that ATP, phosphocreatine, and MgCl2 were omitted and the nuclear extract was preincubated at 30 C for 30 min to deplete any endogenous ATP. Reactions were incubated at 30 C for either 45 min (splicing) or 40 min (E complex) immediately prior to gel filtration. A 500-jui aliquot of either a splicing reaction or an E complex assembly reaction was loaded onto a 1.6 X 50-cm Sephacryl S-500 gel filtration column equilibrated and run in 20 mM Tris (pH 7.8), 0.1 Triton X-100, 60 mM KCl, and...

Preparation Of Tissue

Due to the presence of mineralized matrix within resection and biopsy specimens, routine processing techniques, utilized in soft-tissue specimens, cannot be performed. Bony tissue received within the laboratory is customarily fixed in 10 formalin. If a hematologic, lymphop-roliferative process or other small round blue cell tumor is clinically suspected, the specimen should be sent fresh (without fixative). Tissue can then be harvested for flow cytometric analysis, molecular diagnostics, or cytogenetic studies. Additionally, microbiologic cultures can be performed. A tissue imprint (smear) can provide preliminary information as to adequacy of sample or aid in the diagnosis. A frozen section may be attempted if tissue is deemed adequate and without significant osseous matrix. After formalin fixation, a decalcification step must be performed prior to further processing. This may involve a commercially available acid-based solution (Decal) or an ethylenediaminetetraacetic acid-containing...

Sequential Extraction and Separation

The conventional methods to differentiate organic and inorganic P components in soils basically rely on extraction with alkaline or acid reagents. They go back to the procedure originally developed by Chang and Jackson (1956), later further modified (Hedley et al. 1982 Kuo 1996). Many studies use NaOH, often in combination with chelating agents, such as EDTA, or resins. It is assumed that the chemical extractants sequentially remove dis-cretegroupsofPcompounds. TheinorganicPfractionintheextractisthen determined by a colorimetric procedure and the amount of organic P is obtained by difference (Condron et al. 1985 Guggenberger et al. 1996 Sui et al. 1999 Koopmans et al. 2003). Another approach is to identify and quantify organic P forms with enzymatic mineralisation. The sample is incubated with specific P-releasing enzymes, such as alkaline or acid phosphatases, phytases or nucleases. The orthophosphate released is determined by col-orimetric procedures, most often the molybdate blue...

Interactions of Metal Ions with the pAmyloid Peptide

Earlier studies have shown that metal ions like Cu2+ or Zn2+ may induce very efficient aggregation of soluble Ap. Zn2+ ions induce aggregation at pH 7.4 in vitro and this reaction is reversible with chelation 66,67 , while Cu2+ ions are much more effective in conditions representing physiological acidosis (pH 6.6-6.8) 68 . It was also shown that aggregation is negligible for the rat (mouse) peptide, which differs from the human variant by three substitutions Arg5 Gly, Tyr10 Phe, and His13 Arg. Preliminary data clearly indicated the role of metal interaction with the imidazole donor set of the His side chain in the aggregation process and possible multi-copper binding to Ap 66-68 . Recent studies based on EPR measurements seem to indicate that hAp binds Cu2+ in a mononuclear metal binding site with a metal peptide ratio of 1 1 both in soluble and fibrillar form 69 . The analytical data concerning Alzheimer's plaque formation are extensive although there was not much analytical...

Membrane vesicle preparation

Membrane vesicles (bacterial cell ghosts) have been key tools for the analysis of bacterial membrane proteins since Kaback and colleagues developed techniques for their preparation in the 1960s. To prepare membrane vesicles, cells are plasmolyzed, treated with EDTA and lysozyme to compromise the integrity of the murein layer (releasing periplasmic contents), lysed osmotically to release cytoplasmic contents, harvested, and washed by repeated centrifugation (Kaback, 1971, 1974 Konings, 1977 Konings et al., 1973). Extensive characterization has shown that, correctly prepared, each ghost is derived from a single cell and retains the topology of its cytoplasmic membrane (Altendorf and Staehelin, 1974). However such preparations must be used with care, as topology can be disrupted during vesicle preparation and handling.

Alkaline Gelloading Buffer

300 mM NaOH 6 mM EDTA For a 6x buffer. EDTA To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Isolation and Purification of DNA Fragments for Microinjection

Tris-Borate EDTA buffer (TBE) 89 mM Tris-HCl (pH 8.3), 89 mM boric acid, 2 mM EDTA. Made as a 5X stock solution and diluted to 1X as required with the addition of deionized water (Elga, High Wycombe, Bucks, UK) containing 0.5 pg mL ethidium bromide (from a 10 mg mL stock). 11. Microinjection buffer 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA. Make up using high-quality, endotoxin-free water that has preferably been embryo-tested (e.g.,W1053, Sigma). Sterilize by passage through a 0.22- im filter. Store in 10-mL aliquots at -20 C.

Compleximetric titrations

These titrations are used in the estimation of metal salts. Ethylenediamine tetracetic acid (EDTA) shown in Figure 3.10 is the usual titrant used. It forms stable 1 1 complexes with all metals except alkali metals such as sodium and potassium. The alkaline earth metals such as calcium and magnesium form complexes which are unstable at low pH values and are titrated in ammonium chloride buffer at pH 10. The general equation for the titration is EDTA. The end-point of the reaction is detected using an indicator dye. The dye is added to the metal solution at the start of the titration, and forms a coloured complex with a small amount of the metal. The first drop of excess EDTA causes this complex to break up resulting in a colour change. Titration with EDTA is used in the pharmacopoeial assays of bismuthsubcarbonate, calcium acetate, calcium chloride, calcium gluconate, magnesium carbonate, magnesium hydroxide, magnesium trisilicate, bacitracin zinc, zinc chloride and zinc undecanoate....

CIP Dephosphorylation Buffer

EDTA To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Isolation of Primordial Germ Cells

Tissue-culture reagents can be purchased from Grand Island Biological Company (Gibco, Grand Island, NY, 1-800-828-6686). Trypsinization solution is 0.25 trypsin, 1 mM EDTA in Hank's balanced salts (cat. no. 25200-056). Dulbecco's phosphate-buffered saline (PBS) is 0.2 g L KCl, 0.2 g L KH2PO4, 8 g L NaCl, 1.15 g L Na2HPO4, and 2.16 g L Na2HPO47H2O. (cat. no. 14190). Medium is Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4.5 g L glucose (cat. no. 11965-084). For the culture of PGCs, this medium is further supplemented with 0.01 mM nonessential amino acids (cat. no. 11140-019), 2 mM glutamine (cat. no. 25030-016), 50 pg mL gentamycin (cat. no. 15750-011), 0.1 mM 2-mercaptoethanol (cat. no. 21985-023), and 15 fetal bovine serum (ES cell tested, from either Summit Biotechnology, Fort Collins, CO, 1800-933-0909 or HyClone Laboratories, Logan, UT, 1-800-492-5663). For the routine culture of fibroblast feeder cells, the DMEM with glucose is supplemented with 10 fetal bovine...

Electrophoresis of DNA Samples

The two most commonly used buffers are TAE (0.04 M Tris base, 0.02 M glacial acetic acid, 1 mM EDTA) and TBE (0.089 M Tris base, 0.089 M boric acid, 1 mM EDTA). Although DNA fragments migrate about 10 faster in TAE buffered gels than in TBE gels, the difference in resolution is negligible. Note that the buffering capacity of TAE is low, so the buffer requires replacement or recirculation during extended electrophoresis runs. This step is not necessary when using TBE.

Extraction for Metagenome Libraries

To extract bacterial DNA for the creation of clone libraries (metagenome projects Rondon et al. 2000 Lorenz and Schleper 2002), it is desirable to achieve high molecular weight and unbiased DNA (Lorenz and Schleper 2002 Berry et al. 2003). The necessity of large DNA inserts is particularly important when using vectors which can carry large (100 kb) inserts (BAC, Cosmid) which are used to construct libraries suitable for sequence homology-based screening. Thus, Ginolhac et al. (2004) use a DNA extraction protocol which ensures a minimum of physical DNA shearing cells are first extracted from the soil (on Nycodenz), then mounted in an agarose gel plug prior to lysis (by lysozyme and achromopeptidase) and extraction (100 mM EDTA, 1 lauryl sarcosyl, proteinase K).

Low Calciumlnduced Epileptiform Activity

1982 Yaari et al., 1983) showed that lowering extracellular Ca2+ (i.e., in the bathing medium) could induce regular seizure-like events that lasted for about 30 seconds and recurred in a clock-like fashion. Similar events can also be induced when the hippocampus is treated with Ca2+-chelating agents in vivo (Feng and Durand, 2003). This pattern of activity depends on low (zero mM) Ca2+ treatment and simultaneous elevation of K+ o to 5.5mM. The induction of seizure-like events coincides with the blockade of evoked synaptic transmission. It is a result of reduced surface charge screening (which increases neuronal excitability) and reduced Ca2+-dependent K2+ currents. Electrographically the events appear to mimic tonic-like discharges with slow negative shifts on which a series of population spikes are superimposed. The appearance of population spike activity is somewhat delayed with respect to onset of the negative field potential shift. Clonic-like afterdischarges are not apparent....

Cyclic Alcohol Dehydrogenase Secondary Alcohol Dehydrogenase Membrane Bound

A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction of acetic acid bacteria. After extensive screening, Gluconobacter frateurii CHM 9 was selected. EDTA treatment with the membrane fraction indicated that the membrane-bound cyclic alcohol dehydrogenase is a PQQ-dependent dehydrogenase. From the membrane fraction, PQQ-dependent cyclic alcohol dehydrogenase was purified 31 . In contrast, from the cytoplasmic fraction of the same organism, an NAD-dependent cyclic alcohol dehydrogenase was purified and crystallized. The substrate specificities of the two differently localized enzymes showed an interesting contrast with each other, suggestive of their different physiological roles in the organisms. Unlike the already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, the PQQ-dependent enzyme is unable to catalyze reduction of cyclic ketones or aliphatic ketones to cyclic alcohols or aliphatic secondary alcohols.

Oligodeoxynucleotide Preparation

Oligodeoxynucleotides were synthesized using standard phosphoramidite chemistry. The TMP-F-dU-CE convertible phosphoramidite (Glen Research, Sterling, VA) was used to introduce 5-fluorodeoxycytidine. In certain cases fluorescent labels have been introduced. For this purpose the 5' fluorescein phosphoramidite (Glen Research, Sterling, VA) was used. For experiments in which the DNA Y-junction is to be exposed to cultured cells or crude extracts, the DNA can be synthesized using protective phosphothiolate backbones. These backbones do not interfere with methyltransferase binding but do protect against cleavage by nucleases. However, we have found that this is unnecessary when the devices are used only to target the cell surface. Oligodeoxynucleotide concentrations were measured by absorbance spectroscopy at 260 nm. In order to form duplexes and Y-junctions, oligodeoxynucleotides were mixed in equimolar amounts in a buffer containing 10 mM Tris-HCl at pH 7.2, 1 mM EDTA, and 100 mM NaCl,...

Thermodynamic and Structural Studies of NickelII Complexes of Amino Acids with Side Chain Donors

In the coordination of the nonprotonated form of these ligands the side chain donor plays a crucial role and a tridentate coordination via two joined chelates is expected. Although there is a strong tendency for tridentate coordination with all of these ligands, differences between their nickel(II)-binding ability can also be found. For example, tridentate chelation allows the coordination of two ligands at most to a nickel(II) ion in an octahedral complex. However, this is the case only with Asp and His with Daba, Dapa, and Cys, the situation is more complicated. Although the formation of bis-complexes with tridentate coordination of two Daba or Dapa is favored, some 1 3 complex also appears at high ligand excess 29 . Moreover, with Dapa 29 and Cys 21 , under certain conditions, even a change in the geometry from octahedral to planar occurs. Cysteine does not coordinate nickel(II) in a tridentate way at all, but (S,N)-chelation occurs and a diamagnetic planar 1 2 complex results....

TAFE Gel Conditions for Separating DNAs of Various Sizes Size Range kb Pulse Time Time Hours Seconds

In a Tris-acetate EDTA buffer, a pulse time of 15 seconds will separate DNA fragments in the 50-400-kb size range. This same range of fragments can be separated in the Tris-borate EDTA buffer using a program of 8-second pulses at 350 mA for 12 hours followed by 15-second pulses at 350 mA for an additional 12 hours.

Denaturation Solution

For neutral transfer, double-stranded DNA targets only. EDTA To prepare EDTA at 0.5 M (pH 8.0) Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Postoperative Surveillance

Although most lab tests are not helpful in confirming the diagnosis, chromium EDTA,23 or Technetium DPTA24 isotope studies have been useful in identifying increased intestinal permeability which correlates well with, but is not specific for, rejection. If rejection is suspected, endoscopic evaluation of the intestinal graft must be performed. The endoscopic evaluation should include as much of the small bowel as possible and biopsies from numerous sites (at least 6) should be obtained, since rejection can often be segmental. The loop ileostomy greatly facilitates this type of assessment and for that reason the ileostomy is usually kept in place for 6 months to a year following the transplant. Although the endoscopic appearance of rejecting small bowel is often abnormal with evidence of inflammation and ulceration, in early rejection it can be quite normal. Zoom-endoscopy appears to provide more a valuable endoscopic identification of acute rejection in the small bowel. The gold...

Complexes of Aminohydroxamates

The hydroxamic acid group (which is a weak monoprotonic group with a pKa of about 8-9) is known as one of the most efficient metal ion-binding sites in natural systems 57 . Since both the natural and synthetic hydroxamic acids show numerous biological effects, there has been continuous interest in studies of such compounds. Natural hydroxamate-based compounds, siderophores, play a crucial role in microbial iron(III) uptake, storage, and transport 58 , but the hydroxamic acids are also efficient chelating agents for many other metal ions and are effective inhibitors of metalloenzymes (e.g., nickel(II)-containing urease) 59 .

Reagents and Supplies

Buffer aqueous buffer compatible with the SEC column requirements for the majority of SEC media, 150 mM salts must be present to prevent electrostatic interactions with the column's matrix (for the analysis of a-HL and LamB 20 mM HEPES, 200 mM NaCl, 1 mM ethylenediamine tetraacetic acid EDTA pH 7.4, 5 mM L-glutamic acid, 2 mM (0.05 ) dodecyl maltoside, C12M). The buffer should be filtered through a 0.1- im filter.

Complexes of Oxime Derivatives of Amino Acids

In the oxime derivatives of amino acids the amino group, which is usually an anchoring site for metal ions, is modified hence, this modification is expected to cause a significant change in the coordination ability of the new ligand. According to the results obtained for nickel(II) complexes of several derivatives 70-73 , these ligands are very efficient chelating agents of nickel(II). 2-(Hydroxyimino)propanoic acid (which is the oxime derivative of a-alanine) and its amide, as well as 2-cyano-2-(hydroxyimino)acetic acid and its amide, and also its ethane-1,2-diamine derivatives have been studied (Scheme 3). bond existing between the deprotonated and nondeprotonated hydroxyl groups. Due to the strong electron withdrawing effect of the cyano group, the oxime OH becomes several orders of magnitude more acidic by exchanging the methyl for a cyano substituent in the alanine derivatives. As a result of this effect, the 2-cyano-2-(hydroxyimino)acetate derivatives are somewhat less effective...

Metal chelate affinity chromatography

Metal chelate affinity chromatography is a pseudoaffinity protein purification technique first developed in the 1970s. The mode of adsorption relies upon the formation of weak coordinate bonds between basic groups on a protein surface with metal ions immobilized on chromatographic beads (Figure 6.17). The affinity medium is synthesized by covalent attachment of a metal chelator to the chromatographic bead via a spacer arm. Chelating agents, such as iminodiacetate, are capable of binding a number of metal ions (e.g. Fe, Co, Ni, Cu, Zn, Al), and binding effectively immobilizes the ion on the bead. The affinity gel is normally supplied without bound metal, so the gel can be 'charged' with the metal of choice (by flushing the column with a solution containing a salt of that metal, e.g. CuSO4 in the case of copper). The metal ions most commonly used are Zn2+, Ni2+ and Cu2+. Basic groups on protein surfaces, most notably the side chain of histidine residues,

Complexes of Peptides with Coordinating Side Chains

It has been demonstrated in Section 2 that oxygen, nitrogen and sulfur donor atoms in the side chains of amino acids can significantly influence the metal-binding affinity of the ligands. Among them imidazolyl nitrogen donor atoms of histidyl and thiol sulfur atoms of cysteinyl residues are the most common and most efficient metal-binding sites and their complex formation reactions are discussed in Sections 3.3 and 3.4. In this section we focus on the complex formation of peptides containing alcoholic, phenolate or carboxylate oxygen, lysyl amino nitrogen and thioether or disulfide sulfur atoms in the side chains of natural amino acids. The results obtained on the nickel(II) complexes of various derivatives of peptides including the conjugates of some chelating agents will be discussed as well. The various derivatives of peptides and their metal complexes have received increasing attention in the last decades. These molecules include the insertion of additional functional groups into...

Calcium European Pharmacopoeia 231

A number of anions are mentioned to cause interference. The complex formation can be eliminated completely by the presence of EDTA, oxalate, fluoride, and phosphate, the organic anions through complex formation with calcium, the inorganic anions through precipitation of calcium. Citrate and carbonate, tartrate, and borate also cause disturbances but only when present in high concentrations.

DNA Isolation in Agarose Blocks

Using a bent Pasteur pipet, gently transfer the plugs to a Falcon tube containing the extraction solution (0.5 M EDTA, 1 Sarcosyl, 1 mg mL Proteinase K at least 1 mL per plug). 9. Store plugs in 0.5 M EDTA, pH 8.0, at 4 C until the subsequent steps agarose plugs can be stored in this solution for several years.

Restriction Endonuclease Digestion of DNA in Agarose Blocks

Use 1 4 of plug (corresponding to 0.5 x 106 cells) for each DNA digestion. Cut slices on a sterile surface using a sterile scalpel unused parts can be stored again in 0.5 M EDTA, pH 8.0. 6. The slices may be either used directly or stored in 0.5 M EDTA, pH 8.0 at 4 C for later use.

Arabitol Dehydrogenase Membrane Bound

Dissociation of PQQ was detected by HPLC by SDS-treated ARDH. PQQ and ubiquinone-10 (UQi0) were detected in a purified ARDH when enzyme solubilization was done with dodecyl-P-maltoside instead of Triton X-100. More importantly, when the membrane fraction was treated with 20 mmol L-1 EDTA overnight, ARDH activity was lost but the enzyme activity was restored to its original level by the subsequent addition of PQQ and Ca2+.

DNA Isolation from Lymphocytes

1X Blood lysis buffer (1 L) 155 mM NH4Cl , 10 mM KHCO3, 1 mM EDTA, pH 7.4. This solution should be stored at 4 C up to 6 mo. Blood should be handled using universal precautions. Take care to discard all blood contaminated materials in appropriate biological waste containers. 2. 1X Nucleus lysis buffer (1L) 10 mM Tris-HCl, 400 mM NaCl, 2 mM EDTA, pH 8.2. Store at 4 C up to 1 y. 7. TE-4 (0.5L) 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5. Store at room temperature up to 1 y.

OligodTCellulose Columnloading Buffer

2 mM EDTA (pH 8.0) To prepare the 2x buffer, make up Tris-Cl (pH 7.6) from a fresh bottle in autoclaved, DEPC-treated H2O. Prepare NaCl and EDTA in 0.1 DEPC in H2O. Store for at least 12 hours at 37 C and autoclave the mixture for 15 minutes at 15 psi (1.05 kg cm 2) on liquid cycle. To prepare sterile column-loading buffer, mix appropriate amounts of RNase-free stock solutions of Tris-Cl (pH 7.6), NaCl, and EDTA (pH 8.0) in an RNase-free container. Allow the solution to cool to approx. 65 C, and then add sodium lauryl sarcosinate from a 10 stock solution that has been heated to 65 C for 30 minutes.

OligodTCellulose Elution Buffer

10 mM Tris-Cl (pH 7.6) 1 mM EDTA (pH 8.0) 0.05 SDS The stock solutions of Tris-Cl and EDTA used to make elution buffer should be freshly autoclaved (15 minutes at 15 psi 1.05 kg cm 2 on liquid cycle) and then diluted with the appropriate amount of sterile DEPC-treated H2O. Then add the SDS from a concentrated stock solution (10 or 20 ) made in sterile DEPC-treated H2O. Do not attempt to sterilize elution buffer by autoclaving as it froths excessively.

Sodium complexes of adenosine 5triphosphate

The first crystal structure of an ATP complex, Na2H2ATP3H2O,16-18 was reported by Kennard and coworkers in 1970. Four crystallographically independent Na+ ions and two H2ATP2- molecules exist in the asymmetric unit, where the adenine base is protonated at N1 and the triphosphate group is protonated at a g-phosphate oxygen for each ATP molecule. The sodium ion Na1 (or Na2) binds simultaneously to the base moiety and the phosphate group of the same molecule, thus forming an intramolecular N7-Na-O (g-phosphate of ATP) chelation as shown in Figure 5.2 and Table 5.2. In addition, the Na1 (or Na2) binds to the phosphate group of the neighboring ATP molecule through a-, p-, and g-phosphate oxygens, thus forming a metal ion-bridged dimeric structure, at which a pseudo twofold symmetry exits at the midpoint of the Na1-Na2 vector. The third sodium ion, Na3, is octahe-drally coordinated by an oxygen atom of the p-phosphate from each of two ATP molecules and by four water molecules (not shown in...

Interpatient variability in elimination of anticancer drugs

Hepatic and renal functions are the major determinants of drug elimination and have to be explored before administration of anticancer drugs. Renal dysfunction is easily assessed by determining serum creatinine, measuring or calculating creatinine clearance, or most accurately, by determining the clearance of radio-labelled EDTA. The impact of hepatic dysfunction on the drug elimination or metabolism is more difficult to estimate. Because of complex pathophysio-logical mechanisms of liver insufficiency, hepatic enzymes and serum bilirubin levels are often poor indicators of metabolic activity. Alternative hepatic function tests have limited value in predicting pharmacokinetics of chemotherapeutic drugs

Magnesium and calcium complexes of adenosine 5 triphosphate

Figure 5.2 Dimeric structure of the Na2H2ATP3H2O complex (Kennard, O. et al., Proc. R. Soc. Lond. A, 1971, 325, 401-436), showing coordination of Nal and Na2 ions with the nucleotide molecules Mol. 1 and Mol. 2. Note the intramolecular N7-Na-O(g-phosphate) chelation in addition to the a,P,g-tridentate chelation to the triphosphate group. The same structure is observed in the Na2H2ATP2H2O complex (Sugawara, Y. et al., J. Am. Chem. Soc., 1991, 113, 5440-5445). Broken lines denote interligand hydrogen bonds. Figure 5.2 Dimeric structure of the Na2H2ATP3H2O complex (Kennard, O. et al., Proc. R. Soc. Lond. A, 1971, 325, 401-436), showing coordination of Nal and Na2 ions with the nucleotide molecules Mol. 1 and Mol. 2. Note the intramolecular N7-Na-O(g-phosphate) chelation in addition to the a,P,g-tridentate chelation to the triphosphate group. The same structure is observed in the Na2H2ATP2H2O complex (Sugawara, Y. et al., J. Am. Chem. Soc., 1991, 113, 5440-5445). Broken lines denote...

Uses Of The Polya Polymerase 20methyltransferase

An electrophoretic time-course PAP assay has been described and subsequently refined (Gershon and Moss, 1992, 1993a,b Shuman and Moss, 1988). This assay employs a discrete 5' end-labeled RNA primer, and polyadenylylation products are analyzed by gel electrophoresis. As originally reported (Shuman and Moss, 1988), reaction conditions were similar to those described for a-32P AMP incorporation into trichloroacetic acid (TCA)-precipitable RNA (Gershon and Moss, 1996 Moss et ai, 1975), except for a reduction of the MnCl2 concentration to 0.6 mM, the omission oflabeled ATP, and the use ofa small (10- .1) reaction volume and the RNA primer Ui0 after 5 '-end labeling of the latter using y-32P ATP and T4 polynucleotide kinase. After a time zero sample is removed, reactions are initiated by adding enzyme to a mixture ofthe other components. Aliquots of the reaction mixture are then withdrawn at various times and mixed with equal volumes of deionized formamide. Reaction products are analyzed by...

MOPS Electrophoresis Buffer

0.2 M MOPS (pH 7.0) 20 mM sodium acetate 10 mM EDTA (pH 8.0) For 10x solution Dissolve 41.8 g of MOPS in 700 ml of sterile DEPC-treated H2O. Adjust the pH to 7.0 with 2 N NaOH. Add 20 ml of DEPC-treated 1 M sodium acetate and 20 ml of DEPC-treated 0.5 M EDTA (pH 8.0). Adjust the volume of the solution to 1 liter with DEPC-treated H2O. Sterilize the solution by passing it through a 0.45-pm Millipore filter, and store it at room temperature protected from light. The buffer yellows with age if it is exposed to light or is autoclaved. Straw-colored buffer works well, but darker buffer does not.

Copper and zinc complexes of the type [MH2ATP aromatic amine2

Figure 5.5 Structure of the dimeric Zn(H2ATP)(bpy) 2 complex (Orioli, P. et al., J. Am. Chem. Soc., 1981, 103, 4446-4452), showing the a,P,g-tridentate chelation to the triphosphate group and metal ion-bridged intramolecular face-to-face stacking interaction between the adenine base and the bpy ligand planes. The same coordination mode is observed in the corresponding copper complex (Sheldrick, W.S., Z. Naturforsch, 1982, 37b, 863-871). Figure 5.5 Structure of the dimeric Zn(H2ATP)(bpy) 2 complex (Orioli, P. et al., J. Am. Chem. Soc., 1981, 103, 4446-4452), showing the a,P,g-tridentate chelation to the triphosphate group and metal ion-bridged intramolecular face-to-face stacking interaction between the adenine base and the bpy ligand planes. The same coordination mode is observed in the corresponding copper complex (Sheldrick, W.S., Z. Naturforsch, 1982, 37b, 863-871).

Assays for Antibodies to Glycolipid Antigens

The solid-phase assay has also been adapted for glycolipid antigens (Smolarsky, 1980). The glycolipids are dissolved in ethanol and added to the wells of polyvinyl plates. The ethanol is then evaporated by a stream of nitrogen followed by high vacuum for 5 min, and the plates washed three times in PBS containing 0.3 gelatin and 1 mM ethylenediamine tetra-acetic acid (EDTA). The remainder of the assay is similar to those previously described. Other assays for antibodies to glycolipids have been described by Gray (1979), Young et al. (1979), and Handman and Jarvis (1985).

Molecular conformations of adenosine 5triphosphate

However, a deviation from this rule occurs in ribose puckering and around the exocyclic C4'-C5' bond in Na2'H2ATP2H2O,19 where one of two ATP molecules adopts unusual C4'-endo and trans-gauche conformations. The phosphate chains are fixed in the folded conformation by a, p, g-tridentate chelation in all of the ATP-metal complexes, forming the Pa-Pp-Pg angle of 85 to 100 (Table 5.4). The C4'-O5'-P(a)-O(a,p) chain is conformationally flexible and thus the triphosphate chain points toward the adenine base in Na2 H2ATP1719 or away from the adenine base in M(HATP)2 4- (M Mg2+,21 Ca2+,22 Mn2+,20 Co2+,20 Zn2+,24 and Cd2+(ref.24)), and M(H2ATP)(aromatic amine) 2 (M Cu2+(ref.22) and Zn2+(ref.23)).

Structural types of adenosine 5 triphosphate metal complexes and metal bonding modes

A total of 10 ATP-metal complexes are categorized into three structural types type (1) Na2H2ATP 1719 type (2) M(HATP)2 4- (M Mg2+,21 Ca2+,21 Mn2+,20 Co2+,20 Zn2+,24 and Cd2+(ref.24)) and type (3) M(H2ATP)(aromatic amine) 2 (M Cu2+ 22 and Zn2+(ref23)). In type (1), two kinds of metal bondings are observed one is the simultaneous metal bonding to base and phosphate to form an intramolecular N7-M-O(g-phosphate) chelation and the other is the metal bonding to phosphate only to form an a, p, g-tridentate chelation (Figure 5.2), where the metal ion is bonded more weakly to the O(a) atom than to the O(p) and O(g) atoms (Table 5.3). As noted in Section 5.2.1, metal bonding to the adenine base at N7 may be facilitated by the formation of an interligand hydrogen bond between the amino N6 and a phosphate oxygen (of a different ATP molecule) that ligates to the same metal ion. In type (2), divalent alkaline earth and transition metal ions exhibit phosphate-only metal bonding to a-, p-,...

Single immunoprecipitation

Cells grown on a 100-mm dish are washed with ice-cold PBS and lysed with 0.1 ml of SDS extraction buffer (1 SDS, 50 mM Tris, pH 7.4, 5 mM EDTA, 2 mM PMSF, 20 mg ml aprotinin, 12.5 mg ml pepstatin, and 12.5 mg ml leupeptin) at 4 . The lysate is boiled for 10 min and mixed with 0.9 ml Triton X-100 extraction buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 Trition X-100, 2 mM PMSF, 20 mg ml

Primer extension analysis

High-quality RNA containing abundant mRNA of the target gene is essential. This can be obtained from cells cultured with the optimal hypertonic condition. One or two deoxyoligonucleotides (primers) of 25 to 40 bases complementary to the suspected first exon 50 to 100 bases downstream of the 50 end are made. The primers are end labeled with T4 polynucleotide kinase and purified as described previously for the EMSA probe. The radiolabeled primers are annealed to 10 mg of total RNA or 1 mg of poly(A) RNA in 250 mM KCl, 2 mM EDTA, and 40 mM Tris-Cl, pH 8.0, in a volume of 20 ml. The tube is heated to 95 for 10 min and to 60 for 60 min before being placed on ice. Reverse transcription is performed using Superscript III reverse transcriptase (Invitrogen) using the supplier's instructions except that the reaction is carried out for 1 h at 50 to disrupt secondary structures in mRNA. The extended product is precipitated at -80 after the addition of 0.3 Msodium acetate, 10 mMMgCl2, and 3...

Product case study Ontak

Ontak (tradename, also known as denileukin diftitox) is an engineered fusion protein produced by recombinant means in E. coli. It gained approval for general medical use in the USA in 1999 and is indicated for the treatment of patients with cutaneous T-cell lymphoma (CTCL). The fusion product is composed of diphtheria toxin fragments A and B directly linked to IL-2. It displays a molecular weight of 58 kDa and is purified using reverse-phase chromatography and diafiltration. The product is formulated as a sterile frozen solution containing citric acid, EDTA and polysorbate as excipients. It is presented in single-use vials. The product is normally administered i.v. daily for five consecutive days every 3 weeks over the treatment period.

Analysis for mtDNA Depletion

Make a 1 agarose gel 20 cm in length using 1X Tris-Acetate EDTA (TAE) buffer containing 0.5 g liter ethidium bromide (EtBr). Load the total volume of each digestion product with an appropriate loading buffer in individual wells. In additional wells, load an apropriate molecular weight marker and at least one undigested DNA sample.

Formulations for Nebulization

It is important to profile the solution stability of the drug candidate as early as possible to identify the pH of optimum stability. The influence of light, oxygen, and trace metals on compound degradation also needs to be considered to assess the requirement for antioxidants (sodium metabisulfite, ascorbic acid, etc.) or chelating agents (EDTA, citric acid, etc.). Inclusion of such agents, though required to improve the chemical stability, must be weighed against the potential for adverse effects on the lung. Drug stability in solution should be monitored using a stability-indicating assay, following stress storage as a function of elevated temperature. In addition, conditions that promote hydrolysis (pH extremes), photolysis (ultraviolet and visible light), oxidation (O2, O2 light), and trace metal ion catalysis (FeT , FeT , CuT , CoT , etc.) all merit consideration 3 . Temperature cycling is also useful as a means of assessing potential problems relating to complexation or hydrate...

Mechanisms Of Ocular Penetration Enhancers

As mentioned earlier, tight junctions are the major determinant of paracel-lular transport. In other words, tight junctions are the primary targets for a penetration enhancer to act on in order to improve paracellular transport. The most well-known penetration enhancer to improve paracellular transport is EDTA, which is a calcium chelator commonly used as a preservative. It is well known that proper functioning of tight junctions depends on calcium ions. In the absence of calcium ions, there is a widening of tight junctions, resulting in an increase in paracellular permeability (8). EDTA can remove divalent ions by its chelating action. Therefore, there is no surprise that it has a permeabilizing effect on biological membranes (16). However, its action on the cornea is believed to be much more complicated. Rojanasakul et al. (17) showed that severe membrane damage is evident in corneas treated with EDTA, bile salts, and surfactants. This disruption of plasma membrane structures by...

Nucleic Acids and Oligonucleotides

Anchoring 3' oligonucleotide primers (300 Mg ml) in 10 mM Tris-Cl (pH 7.6), 0.1 mM EDTA The anchoring 3' oligonucleotides are a family of 12 primers with the general structure 5'-d(T)12VN-3', where V is either C, A, or G, and N is C, T, A, or G. For example, one primer in the series is 5'-d(T)12CC-3', the next is 5'-d(T)12CT-3', etc. Arbitrary 5' oligodeoxynucleotide primers (50 Mg ml) in 10 mM Tris-Cl (pH 7.6), 0.1 mM EDTA Sixteen arbitrary 5' oligonucleotide primers are required, each ten nucleotides in length. The sequence of each primer is chosen at random, but it should contain approximately equal numbers of the four bases, with a balanced distribution of G and C residues, and a low propensity to form stable secondary structures.

Telomeric Repeat Amplification Protocol

EDTA EDTA, ethylenediaminetetraacetic acid EGTA, acid. EDTA, ethylenediaminetetraacetic acid EGTA, acid. b. Before use, make up the working solution as follows 4 L of 0.1 M phenylmethyl sulfonyl fluoride (17.4 mg mL in 2-propanol), 1.4 L -mer-captoethanol, 0.84 L of 0.12 M sodium deoxycholate, 10 L NP-40, 40 L RNase inhibitor (10 U L). Bring to 4 mL with NP40 stock buffer. 10X Phosphate-buffered saline (PBS) 80 g NaCl, 2 g KCl, 11.5 g Na2HPO47H2O, 2 g KH2PO4, pH 7.3, to 1 L with sterile H2O. 10 Polyacrylamide stock in 400 mL of 0.5X TBE 100 mL of 40 polyacrlamide solution (19 1), 40 mL of 5X TBE buffer, 260 mL deionized H2O. Final vol 400 mL. 50 mL of 10 Polyacrylamide gel 49.5 mL of 10 polyacrylamide stock (19 1), 0.5 mL of 10 ammonium persulfate, 0.05 mL TEMED. Final vol 50 mL. 50X TAE buffer 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL of 0.5 M EDTA, pH 8.0. Add sterile H2O to 1 L and autoclave. 5X TBE buffer 54 g Tris base, 27.5 g boric acid, 20 mL of 0.5 M EDTA. Add...

Carboxyhydrate Amines A Strong Primary Binding Site May Help in Hydroxyl Group Coordination

In a circular dichroism study of Ni2+ complexes in solution with -glycosylamine ligands formed by 1,3-diaminopropane and pentoses such as d--xylose, D-ribose or D-arabinose, it was concluded that two of the tridentate -glycosylamine ligands coordinate to one Ni2+, each ligand being bound in a meridional mode by the primary amino group, the N-glycosidic secondary amino group and the C2 hydroxyl group of the sugar moiety 37 . For the D-ribose derivative this binding mode was confirmed by an X-ray structure analysis 37 . Interestingly, crystalline Ni2+ complexes of dianionic glycopyranoside ligands were obtained by reaction of Ni tris(2-aminoethyl)amine (OH)2 with methyl-D-glucopyranoside or sucrose 38 . In the latter case (C2)O and (C3)O chelation occurs in the glucose part of the disaccharide. It is important to note that here deprotonated hydroxyl groups participate in Ni2+ binding. In this context it may also be mentioned that Ni2+ complexes of N,N'-alkylated ethylenediamine are able...

Multifunctional Approach Polymeric Penetration Enhancers

Reduction of mucus on the microvillus and dilatation of the intercellular space was also observed 5-10 minutes after administration of poly-acrylic acid gel into the rat rectum. However, these changes were reversible and returned to normal relatively soon. It was not likely that polyacrylic acid gel enhanced penetration solely by its detergent action since it inhibited rather than induced hemolysis, which is commonly observed with surfactants. Another possible mechanism is related to its chelating activity. Polycarbopol and other polyacrylic acid-based polymers are able to chelate calcium (56), which is an essential component for proper functioning of tight junctions. In addition, chelation of cations that are essential for normal activity of enzymes further improves bioavailability. However, this inhibitory effect may be too weak to account for the improved bioavailability (57). In the case of ocular drug delivery, there is reported to be only a minimum amount of metabolizing...

In Vitro Histone premRNA Processing Reaction

Processing in vitro occurs in the absence of divalent ions, and inclusion of high concentrations of EDTA suppresses nuclease activity in the extract. The temperature of incubation and the extract concentration have been optimized for the mouse extracts. An example of a processing reaction is shown in Figure 4. Over 60 of the input pre-mRNA is processed (Fig. 4, lane 2). Processing is dependent on the stem loop, is greatly reduced by including a 30-mer RNA encoding the stem-loop (Fig. 4, lane 3), and is abolished by an antisense oligonucleotide against U7 snRNA (Fig. 4, lane 4). Heating the extract to 50 C also abolishes processing. 1. Set up a reaction on ice in a final volume of 20 fjl containing 2500 counts per minute (0.33 pmol) of 32P-labeled pre-mRNA substrate, 20 mM EDTA pH 8, and 17 u.1 of nuclear extract. Mix by gentle pipetting.

Preparation of Fetal Fibroblasts

From this stage on each carcass is treated separately to generate its own primary culture. Transfer individual carcasses to 5 mL of trypsin-EDTA solution. Facilitate isolation of cells by cutting to approx 1-mm cubes with scissors. This process should be completed in approx 10 min.

Use of Monoclonal Antibodies as Probes for Protein Conformation

Antibodies are very sensitive to protein conformation, and monoclonal antibodies are therefore well-suited to probe conformational changes in proteins. This is illustrated in the case of monoclonal antibody 4H4, which was raised against a putative 'EF Hand' calcium-binding region of the PC-1 antigen (Belli et al., 1994). The EF Hand region from human PC-1 was subcloned into the bacterial expression vector pGEX-KT (Hakes and Dixon, 1992), and used to immunize mice. Monoclonal antibodies were screened on methanol-fixed mouse L cells that had been transfected with human PC-1. A monoclonal antibody (4H4) which reacted with methanol-fixed cells did not stain intact unfixed cells, suggesting that it recognized only an unfolded conformation of PC-1. However, the 4H4 antibody did stain PC-1 on intact living cells in the presence of EDTA. This result suggests that the release of calcium from the EF hand caused a conformational change that allowed the antibody to bind (Belli et al., 1994). In...

Skin Elimination And Reactivity

In general, the divalent cations magnesium and calcium are necessary for intercellular adhesion. In the skin they maintain structural integrity between dermis and epidermis, and within the epidermis itself. The role of magnesium in maintaining cellular adhesion and preventing epidermolysis was demonstrated in mouse skin, when an EDTA solution, routinely used to separate various layers of the skin, failed to produce the desired epidermolysis in the presence of Mg2+, and further, when loss of adhesion induced by EDTA could be reversed by addition of magnesium to the system. The fact that under physiological conditions EGTA (ethylene glycol-bis(P-aminoethyl ether)-N,N,W,W-tetraacetic acid), a strong calcium chelator but weak magnesium chelator, failed to produce epidermolysis the way EDTA did is presented as evidence that magnesium, rather than calcium, is needed for intraepidermal and dermal-epidermal integrity (14).

Preparation of total cell lysates

3T3-L1 adipocytes are serum starved overnight in DMEM 0.5 BSA. 3T3-L1 adipocytes are incubated in serum-free medium supplemented or not with 600 mM sorbitol and subsequently treated with or without a low concentration of insulin (0.2 nM). To study the effect of pharmacological inhibitors, cells are pretreated for 30 min with various inhibitors in serumfree medium followed by incubation in serum-free medium without or with 600 mM sorbitol and pharmacological inhibitors. Cells are subsequently washed with ice-cold buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 10 mM Na4P2O7, and 2 mM sodium orthovanadate) before solubilization for 30 min at 4 in lysis buffer containing phosphatase and protease inhibitors to keep on the level of phosphorylation of proteins (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 150 mM NaF, 2 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride PMSF , protease inhibitors cocktail, 100 nM okadaic acid, and 1 Triton X-100). Clarified lysates...

Laboratory Issues

Various sources of DNA are acceptable for testing. Ethylene diamine tetra-acetic acid (EDTA) anticoagulated peripheral blood is the most commonly submitted specimen however, DNA, frozen tissue, or cultured cells also may be used. Paraffin-embedded tissue, cheek epithelial cells, unspun amniocytes, or direct chorionic villus sampling (CVS) specimens may be used for detecting point mutations by DNA sequence analysis however, these samples do not provide sufficient quantities of high-molecular-weight genomic DNA for Southern blot analysis.

Recirculated alkaline wash

This step uses a hot alkaline solution to solubilize the remaining proteins and other contaminants. A typical composition of this solution is sodium hydroxide, potassium hydroxide or a mixture thereof at a concentration of 0.5-3 in deionized water depending on the water quality and 'soil', species such as chelating agents (EDTA), surfactants or sodium hypochlorite (30-100 ppm) should be added. Temperature should be in the range of 60-85 C. Exposure time for efficient cleaning is very variable (10 min-1 h) depending on the 'soil' and process circuit recirculation of the alkaline solution is thus recommended for economic reasons.

Structure of a Metal Substituted Urease

Since the discovery that urease, a hydrolytic enzyme, contains Ni2+ as the essential cofactor 12 , the question about the role of this metal ion in the catalytic mechanism has intrigued the bioinorganic chemistry community. An approach to the solution of this problem has involved attempts to substitute the essential element with other ions such as Zn2+, Co2+, and Mn2+. Removal of both Ni2+ ions by treating JBU with EDTA at low pH causes irreversible denaturation of the protein 64 . Removal of a single Ni2+ ion by dialysis in the presence of citrate was reported 65 , indicating that the two metal ions are bound with different affinity to the protein matrix. This result can now be interpreted on the basis of the crystal structures of KAU, BPU and HPU, which show that Ni(1) is bound to three protein residues (two histidines and one O-atom of the carbamylated lysine), while Ni(2) is additionally

Acid detergent lignin

NDF content is determined by extracting a 0.5-g sample in boiling neutral detergent solution (3 (w v) sodium dodecyl sulfate (SDS sodium lauryl sulfate)), 18 mM sodium tetraborate decahydrate (Na2B4O7 .10 H2O), 32 mM sodium phosphate (dibasic N2HPO4), 50 mM Na2-EDTA, and 1 (v v) ethylene glycol (2-ethoxyethanol) for 1 h. The suspension is filtered, and the cell wall residue on the filter is washed in hot water and acetone. After drying overnight at 100 C the sample is weighed.

Determinants of gut permeability

The concept that the gut may cause systemic illness and remote organ injury dates from the end of the nineteenth century, and more recently phrases such as the gut being 'the motor of multiple organ failure' and 'the largest undrained abscess in the body' have established the theory in the medical imagination. Permeability of the gut mucosa has been studied using various molecular probes such as sugars or 51Cr-EDTA. Increased permeability has been demonstrated following many different physiological insults including ischemia-reperfusion injury, endotoxemia, systemic acidoses, glutamine deficiency, and certain cytokines such as g-interferon. One of several possible mechanisms for hyperpermeability is increased synthesis of nitric oxide by gut epithelium, neutrophils, or vascular endothelium. Nitric oxide may exert different effects on permeability depending on the nature of the stimulus, its timing, and the experimental model being used these factors may explain why inhibition of...

Molecular Markers Of Tumor Recurrence Or Progression

Tallini, et al. (67) subsequently reported data using different RT-PCR assays for detection of thyroid transcripts from peripheral blood. In this study, the investigators evaluated 44 patients including 24 with thyroid cancer (16 with metastases and 8 free of disease), either pre-operatively, postoperatively, or at both time points for peripheral blood expression of thyroglobulin, thyroid peroxidase, and the RET PTC1 thyroid oncogene. 56 of the patients with either local or distant metastases had positive assays, compared to 63 of those thought to be free of disease. Of those thought to be free of disease that had positive assays, 80 had cervical adenopathy at diagnosis and were felt to be at high risk of tumor recurrence. Of the patients with benign disease, 2 of 20 patients had a positive mRNA assay, both of which reverted to negative after surgery. The in vitro sensitivities of this assay were approximately 50 cells ml of blood. Technically, these authors isolated total RNA from...

The Optical Mapping System

Traditional procedures for DNA extraction involve pipetting, phenol extraction, and centrifugation. During these steps, the DNA molecules are damaged by mechanical shearing forces, and as a result are generally under 100kb. To get longer DNA molecules (> 500 kb), Schwartz and Cantor (1984) developed a method to isolate intact genomic DNAs for pulsed field gel electrophoresis (PFGE). Cells of any type can be embedded in low melting point agarose, a matrix that traps the DNA molecules, yet facilitates diffusion of large macromolecules (average pore size 100nm). Such immobilization allows for successive enzymatic treatment steps, while also offering protection from mechanical shearing and nuclease degradation. For microbes and plants, cell walls are first digested with protoplasting enzyme cocktails. For mammalian cells, this step is not necessary. Next, cells are completely lysed using a combination of N-laurylsarcosine and proteinase K in the presence of EDTA (0.5 M, pH 9.5). The...

Ternary complex formation

The incoming nucleotide also brings in two divalent metal ions, one catalytic and the other for nucleotide binding, to coordinate the oxygen ions of the triphosphate of the nucleotide the side chains of strictly conserved Asp185 and Asp110 and the backbone carbonyl oxygen of Val111. The metal ions are positioned closely to the 3'-hydroxyl group of the primer terminus. The conformational change induced upon nucleotide binding specifically positions Asp110 for metal chelation. The nucleotide base stacks against the primer terminus and residues Arg72 and Gln151. The triphosphate portion

Drug Efficacy in Animal Models of Zygomycosis

Another study evaluated the effect of deferiprone, an iron chelator, in a mouse model of R. oryzae infection (Ibrahim et al., 2006). Treatment with deferiprone increased survival rate and reduced fungal burden in brain compared to untreated controls with an efficacy similar to that of liposomal amphotericin B given at 15 mg kg day. Administration of free iron was shown to suppress the effect of deferiprone therapy demonstrating that the mechanism of protection was chelation of iron (Ibrahim et al., 2006).

New Treatment Strategies in Patients

Despite antifungal therapy, mortality rate in zygomycosis remains very high and there is a need for new therapeutic strategies. Iron chelation could be one of these strategies. Very recently, deferasirox, an iron chelator approved for treatment of iron overload has been successfully used as salvage therapy in a patient with rhi-nocerebral zygomycosis (Reed et al., 2006).

Select Stably Transfected Cells

We save stably transfected cells by freezing them in liquid nitrogen. To do so, cells are treated with Trypsin-EDTA medium and then collected by centrifugation. The cells are then resuspended in the freezing medium at 3-5 x 106 cells mL. The cells are first placed in a -80 C freezer for overnight or a couple of wk. For a long-term storage, the cells are transferred from the -80 C freezer to a liquid nitrogen tank (see Note 5).

Could Antioxidant Agents Prevent the Deleterious Consequences of Oxidative Stress

There are various types of antioxidant compounds direct, indirect, and metabolic. Direct antioxidants have the potential to directly interact with ROS. Their activity is not dependent on endogenous cellular macromole-cules (e.g., enzymes) to exert their primary action but they can by themselves chemically react with the damaging free radicals at the molecular level. Nevertheless, to some extent, there is also interaction with intracellu-lar enzymes since some of these compounds are presumed to be recycled either by endogenous oxireductases directly or indirectly via intracellular reducing shuttles such as ascorbate or some thiols. This class of antioxi-dants include, among others, vitamins, Gingko biloba (flavonoids and terpenoids), and -acetylcysteine. Indirect antioxidants are compounds that do not have antioxidant capacity per se but help to prevent reduce free radicals formation. Among these, we can find metal-chelating agents such as DFO and clioquinol. Metabolic antioxidants are...

Product case study Bene Fix

At least one monoclonal antibody has been raised which specifically binds only to factor IX which contains pre-bound Ca2+ (i.e. the Ca2+-dependent conformation of factor IX). Immobilization of this antibody allowed the development of an immunoaffinity system in which factor IX binds to the column in the presence of a Ca2+-containing buffer. Subsequent elu-tion is promoted simply by inclusion of a chelating agent (e.g. EDTA) in the elution buffer.

Immunosuppression During Pregnancy

Food and Drug Administration (FDA) categorizes the potential fetal risks of drugs using the following classification system A controlled studies, no risk B no evidence of risk in humans C risks cannot be ruled out D positive evidence of risk X contraindicated. The commonly used immunosuppressive agents are listed in Table 21.1 none of these drugs is Category A, the corticosteroids and basiliximab are Category B, and most are Category C. In infants born to renal transplant recipients exposed to azathioprine (Category D), two early reports described the incidence of congenital anomalies as 9 and 6.4 respectively.2,3 There was, however, no specific pattern noted among the kinds of anomalies that occurred. More recent reports have been more reassuring. The extensive European Dialysis and Transplant Association (EDTA) report on 490 pregnancies (500 babies) concluded that azathioprine and prednisone immuno-suppression was not associated with more congenital malformations in the...

Indirect Antioxidants

Recently, a novel system of chelation therapy through the use of nanoparticles has been proposed (Liu et al., 2005). Nanoparticles conjugated to chelators show the ability to cross the blood-brain barrier, chelate metals, and exit through the blood-brain barrier with their corresponding complexed metal ions. The authors suggest that this method may prove to be a safe and effective means of reducing the metal load in neural tissue thus staving off the harmful effects of oxidative damage and its sequelae (Liu et al., 2005). However, clinical studies based in larger sample sizes and longer study periods should be performed.

Strategies For Future Studies

Although I, Re, Re, and Lu are ideal radionuclides for external single-photon imaging, surrogate gamma emitters must be used to evaluate the distribution and clearance of pure beta emitters. 111In has been considered to be an appropriate surrogate for 90Y. Their half-lives are almost identical, and both are readily incorporated into the same metal-chelating agents. A recent study using PET imaging to compare 86Y and 111In as surrogates for 90Y showed that, although 111In and 86Y have similar biodistribution, 86Y remained in organs, such as bone for a longer period of time (97). 86Y is a more suitable surrogate for 90Y, and the short T1 2 of

Urinary Tract Infection In Children

Therefore, the rationale for DMSA scans is likely to change, and they will be done to identify the kidney at risk of progressive deterioration. In this scenario, my personal view is that we will then change to assessing the single kidney glomerular filtration rate (SKGFR), using the DMSA to look for further focal renal damage as well as the divided function, together with the total GFR from a single-sample Cr-51-ethylenediamine tetraacetic acid (Cr-51-EDTA) clearance performed at the same attendance. From the divided function and the total GFR, the SKGFR may be calculated. This is a practical combination (already used in some centers). The child attends, and is cannulated. The Cr-51-EDTA is injected, followed by the DMSA. The child returns three to four hours later when the sample is taken for the GFR estimation, the cannula is removed, and the DMSA images are recorded.

Clinical aspects and treatment of ALAS2 deficiency

Although the anemia of pyridoxine-responsive SA is treatable, the main complication of the disease is iron overload if left untreated, it has the same deleterious results as hereditary hemochromatosis. Iron overload can be biochemically evident as early as in adolescence, does not correlate with the degree of anemia, and can affect mildly anemic females. The importance of effectively treating iron overload cannot be overemphasized for one further reason excess iron interferes with the function of ALAS2 and patients previously unresponsive to pyridoxine, after effective iron chelation, occasionally become responsive.

Subtractive Hybridization

Prepare first-stranded target cDNA (see Note 17) from poly (A)+ mRNA as described above (preferred) or in vitro sense RNA derived from a target cDNA library. In the latter case one should use 5 pg of random hexamers as primers, since one cannot be sure that the poly A tail is present after XhoI digestion. Remove the RNA by adding 10 pL of 0.25 M EDTA and 30 pL 0.15 N NaOH, and incubating for 60 min at 65 C. Neutralize with 30 pL of 0.15 N HCl, ethanol-precipitate, rinse, and dry. 4. Short hybridization spin an appropriate amount of biotinylated driver RNA precipitate (10-fold molar excess to the cDNA) rinse and dry it. Resuspend the RNA in 20 pL of hybridization buffer (HBS 50 mM HEPES, pH 7.6, 0.2 SDS, 2 mM EDTA, 500 mM NaCl).

Materials for Entire Class

96.8 g of Tris base + 22.87 ml of glacial acetic acid + 0.75 g EDTA. Add distilled water to 1 liter. Mix well. Store at room temperature. Mix 4 g of HindIII-cut X DNA with 10 l of loading solution and bring the total volume to 100 l with DNA buffer (10 mM Tris + 0.1 mM EDTA). Use 10 l lane (400 ng lane).

Magnesium and alkalineearth metals European Pharmacopoeia 247

The test makes use of a complexiometric titration with the chelator ethylenediamine tetra-acetic acid (EDTA) using Eriochrome Black as a met-allochromic indicator. EDTA (Figure 6.7.1) forms stable complexes with nearly all polyvalent metal cations and even some monovalent too. It is a multidentate complex binder capable of acting as six ligands on the same central ion (Figure 6.7.2). Figure 6.7.1 Ethylenediamine tetra-acetic acid (EDTA). Figure 6.7.1 Ethylenediamine tetra-acetic acid (EDTA). Figure 6.7.2 EDTA calcium complex. This stability constant deals only with the species of EDTA where all car-boxylic acid groups are deprotonated. The pKa values for these groups are pKa1 2.0, pKa2 2.67, pKa3 6.16, and pKa4 10.3, so the fully deprotonated EDTA dominates only in very alkaline solutions with pH above 12. In solutions of lower pH one has to calculate Y4- at the actual pH and use this concentration in calculating a stability constant. Similarly, if the metal M participates its other...

Pmpa And 2mppa Are Potent And Specific Gcp Ii Inhibitors

Research at Guilford has focused on the potential utility of GCP II inhibitors wherein excess glutamate neurotransmission has been implicated. 2-(Phosphonomethyl) pentanedioic acid (2-PMPA) is a potent, competitive GCP II inhibitor with a Ki value of 0.2 nM15 16 (Fig 2). It exhibits a fast association rate and a slow dissociation rate. The high potency of 2-PMPA can be attributed to the strong chelation of the phosphonate group to an active site zinc atom as well as the interaction of the glutamate moiety (pentanedioic) portion of the inhibitor with the glutamate recognition site of GCPII.18 2-PMPA seems to be quite specific for GCP II, i.e., no significant activities were observed at 10 j.M (more than 10,000-fold higher than the Ki for NAALADase inhibition) in over 100 different receptor and enzyme assays, including glutamate receptors and

Diagnosis of congenital infection

In newborns suspected of having congenital infection, cord blood or and EDTA blood could be collected and analysed for the parasite itself or its DNA. Furthermore, it is recommended that the antibody response be followed by collecting blood samples at birth and at the age of 3, 6 and 12 months to measure the IgG, IgM and IgA antibodies.

Hematological screening

In the measurement of red cells there are significant differences in the analyses carried out by different automated cell counters. While the red cell count will often be similar, the red cell indices, particularly the mean cell volume, may vary substantially. As a measure of cell size, the mean cell volume has considerable drawbacks. These arise from the essentially elastic nature of the red cell. In vivo, red cells do not have a fixed volume. Ex vivo, their volume will vary in relation to storage conditions and oxygenation. A highly agitated and oxygenated sample will have a lower mean cell volume than an unoxygenated sample, but as time passes the cells in an EDTA anticoagulated sample will swell as they take up water. In addition, the red cell can function as an osmometer and will reflect, for example, changes in D-glucose plasma levels. In contrast, the mean cell hemoglobin is unaffected by different technologies and totally unaffected by the storage changes which afflict the...

Mechanism Of Peptide Transport

Minor inflammation of the cornea and calcium chelation may cause a widening of the intercellular spaces, thereby allowing a substantial flux of the peptide compounds. If polypeptide absorption in therapeutic amounts across the cornea is to be achieved, it will be necessary to maintain prolonged contact of the peptide with the corneal surface and also to employ a proper penetration enhancer to potentiate flux across the intercellular spaces.

Postantibody Washes and Histochemistry

For in situ hybridizations using a single probe, stop the reaction by washing thoroughly in PBT containing 20 mM EDTA or in 1X TBST. Refix in 4 paraformaldehyde prior to storing at 4 C or sectioning. Embryos can also be stored at 4 C in autoclaved 90 v v glycerol, 1X PBS.

Estimation of Renal Function

Renal function is usually assessed through calculation of glomerular filtration rate (GFR). The reference method for estimating GFR is inulin clearance. Inulin is an inert polysaccharide cleared exclusively by glomerular filtration. The method includes constant intravenous infusion of inulin and timed collection of urine and is not practical for routine clinical purposes. A number of alternative methods have been developed for estimation of GFR. Many involve collection of urine and may give inaccurate results unless collection of urine is complete, including complete emptying of the bladder. Several methods to determine the plasma clearance of a suitable exogenous marker have been developed. These include radionuclides such as 51Cr-EDTA and 99mTc-DTPA (diethylenetriaminepentaacetic acid) 21 . Although these methods are accurate, the requirement of radiolabeled tracers complicates the procedure (complicated handling, storage, and disposal of waste) and excludes certain patients, such...

Purification of Expressed Proteins from Inclusion Bodies

The expression of foreign proteins at high levels in E. coli often results in the formation of inclusion bodies composed of insoluble aggregates of the expressed protein. The inclusion bodies are recovered from bacterial lysates by centrifugation and are washed with Triton X-100 and EDTA to remove as much bacterial protein as possible from the aggregated foreign protein.To obtain soluble protein, the washed inclusion bodies are dissolved in denaturing agents and the released protein is then refolded by gradual removal of the denaturing reagents by dilution or dialysis. The procedure given here has been used to solubilize prorennin inclusion bodies. However, each protein may require a slightly different procedure, which must be determined empirically.

Aquaporin function in plant membranes

Following the procedure of Kjellbom and Larsson (1984), the first step of preparing plasma membranes is the isolation of crude membrane fractions, the microsomes. Starting material can be leaf or root tissue. One hundred grams fresh weight is homogenized three times for 30 s in 300 ml 0.33 M sucrose, 50 mMHEPES KOH, pH 7.5, 5 mMEDTA, 5 mMdithiothreitol (DTT), 5 mM ascorbic acid, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 bovine serum albumin (BSA), 0.2 casein (boiled for 10 min), and 0.6 polyvinylpolypyrrolidone (to remove phenolic compounds) using a kitchen homogenizer. The homogenate is filtered through three layers of miracloth, and the bulk of chloroplasts and mitochondria is sedimented by centrifuga-tion at 5000 for 10 min. The membranes in the supernatant are collected at 100,000 for 1 h. This pellet is resuspended to a total volume of 10 ml in 0.33 M sucrose, 5 mM HEPES KOH, pH 7.5, 5 mM KCl, 1 mM DTT, and 0.1 mM EDTA. The two-phase partitioning system (Lundborg et al., 1981...

The Medicinal Chemistry Of Metalcentered Brain Disorders

Drugs like penicillamine, trientine, ethylenediaminetetracetic acid (EDTA), and desferrioxamine are useful in the treatment of systemic disorders of toxico-logical metal exposure or genetic disorders (e.g., Wilson's disease) where metals accumulate in tissue. These drugs are the agents utilized in 'chelation therapy', a term associated with the removal of bulk metals such as in Wilson's disease (Cu) CQ was given orally in a blinded study to Tg2576 transgenic mice 4 . The results showed a 49 decrease in brain Ap burden compared with nontreated controls after 9 weeks, with no evidence of toxicity, and the general health and body weight parameters were more stable in the treated animals. Treatment with CQ did not lead to a systematic decrease in metal levels, most likely due to the drug's moderate binding affinities. Interestingly, in the same study, TETA ( trien, trientine), a hydrophilic high-affinity metal chelator that is incapable of crossing the BBB, and has been used to treat...

Presequence Dependent Import

Minor band of 17 kDa (precursor protein) and a major band of around 15 kDa (mature protein), consistent with the removal of the IscU amino-terminal presequence. The cleavage of IscU and ferredoxin targeting signals has also been observed in recombinant parasites that overexpress GFP fusion proteins containing the corresponding targeting presequences at the amino terminus. In these cells, precursor GFP proteins accumulate in the cytosol, whereas mature proteins are found exclusively in the organellar fraction (Regoes et al. 2005). Such accumulation of precursor protein in the cell cytosol demonstrated that mitosome import, like mitochondrial and hydro-gensomal import, is an active and saturable process. Further evidence for the proteolytic processing of giardial IscU presequence comes from in vitro experiments in which radiolabelled IscU was incubated with mitochondrial and hydrogensomal lysates (Dolezal et al. 2005). In these experiments, the targeting presequence is cleaved off in a...

Affinity Purification of Antibody Fragments from coli Supernatant

Many laboratories now include hexahistidine tags in their scFv constructs to aid purification on chelation columns. This approach has the advantage that binding to the column can be performed under denaturing conditions, if necessary. This may be useful if the antibody must be denatured before it can be recovered in a water-soluble form. Alternatively, one can use immunoaffinity chromatography, which must be performed under native conditions.

Advantages of Hydroxyl Radical Footprinting

The hydroxyl radical footprinting technique has several advantages (Shafer etai, 1989). Nucleic acid strand scission is mediated by hydroxyl radicals (Pogozelski et al, 1995, and references therein) generated by the reduction of hydrogen peroxide (H202) or dioxygen (02) by iron(II) (Bull et al., 1983 Tullius and Dombroski, 1986). Since all sugar moieties accessible to solvent (Hertzberg and Dervan, 1984 Wu et al., 1983 Wu and Kozarich, 1985) are the target of attack by these small, diffusible radicals (Pogozelski et al., 1995, and references therein), minimal specificity (Shafer et al., 1989) for either nucleotide sequence (Henner et al., 1982 Hertzberg and Dervan, 1984 Tullius and Dombroski, 1985) or secondary structure (Celander and Cech, 1990) is evident. Due to these properties, the hydroxyl radical is an excellent probe for a high-resolution analysis of backbone regions protected by specific nucleic acid-protein interactions (Tullius and Dombroski, 1986 Wang and Padgett, 1989)....