1. A 20-pmol aqueous solution of PMSF at pH 8.0 has a half-life of 35 min, and therefore, this inhibitor must be added just prior to use (12). The inclusion of 10 mM iodoacetamide is also useful to prevent crosslinking of proteins owing to the presence of free sulfhydryl groups during immunoprecipitation.
2. The dissociation buffer is usually made reducing by the addition of 0.1 M dithiothreitol or 1.4 M P-mercaptoethanol prior to boiling, under which conditions disulfide-linked chains will become dissociated. Such reagents maybe omitted, and this may be useful in immunoprecipitation techniques where the immunoreactive Ig chains in their reduced form (25 and 50 kDa) obscure the antigen in question.
3. The way cells are lysed has a critical effect on the composition of the extract (13,14). Nonionic detergents, such as Nonident P-40 (NP-40), solubilize antigens in their native state and preserve the nuclei in an intact state, so that they may be removed by centrifugation. Mild detergents such as CHAPS, digitonin, and Brij 96, enable macromolecular complexes to be immunoprecipitated allowing protein associations to be investigated. However, the most widely used and versatile immunopre-cipitation buffer is RIPA. This lysis buffer is often the best to use when little is known about the solubilization behavior of the antigen in question.
4. Strong ionic detergents, such as SDS, denature the antigen, and so can be used with antibodies that are considered to be "nonimmunoprecipitating" antibodies.
Such antibodies often include antipeptide antibodies, since they are usually generated against linear epitopes, which may be masked by the tertiary structure of the native protein. Thus, SDS denaturation of the protein allows the masked epitope to be exposed and accessible to the antibody: to do this, lyse the cells in 10 vol of 2% SDS, 50 mM Tris, pH 7.5, and place in a boiling water bath for 10 min. The DNA is then sheared by passing through a 21-gage needle several times, or by using a probe sonicator with several short bursts. Pellet the insoluble material by centrifugation at 10,000g for 10 min, and then dilute the supernatant 20-fold in PBS containing 2% (w/v) BSA.
5. Antibodies or antiserum can be added directly to the cell lysate at 4°C for 1 h or overnight. It is often worth trying both procedures, since the former usually leads to cleaner immunoprecipitates, but if the antibody is of low affinity, the latter may be necessary to achieve optimum binding to antigen. The antibody, including the antibody/antigen complexes; is then recovered using a second antibody against the first, although it is far more usual to use protein A or protein G linked to Sepharose beads. These reagents provide high-affinity binding to the Fc component of the antibody, and make washing and recovery of the antigen very easy (see Note 11).
6. Most MAbs or polyclonal antibodies used in immunodetection procedures are either mouse or rabbit antibodies. With the exception of mouse IgG subclass 1, all these antibodies can be coupled to protein A-sepharose, allowing maximal correct orientation of the antibody for immunoprecipitation. The use of the crosslinker dimethyl pimelimidate (15) also means that the antibody remains attached to the protein A beads during electrophoresis producing "cleaner" Western blots.
7. The percentage attachment of the antibody can be determined by measuring the optical density at 280 nm before and after coupling. If the coupling is low, then either protein G-sepharose or high-salt coupling can be used; for high-salt coupling, the first step of the low-salt coupling procedure is replaced by washing the protein A-sepharose beads three times in PBS, 3 M NaCl, pH 9.0, and then add the antibody for 2 h at 2 mg/mL in the same buffer.
8. Immunoprecipitation can also be used in combination with radiolabeled compounds in order to study protein modifications, such as phosphorylation, or the study of newly synthesized proteins (by 35S-methionine incorporation). The addition of radioisotopes to cell-culture media to follow protein modification of embryonic cell lines is routinely performed, and could be adapted to incubating, over short time spans, early embryos or dissected tissues from fetuses. Experiments have been performed on early embryos for 32P (16), and 35S-methionine labeling of proteins (17). Microinjection of radiolabeled compounds into embryos has also been tried, for example, the labeling of proteoglycans with [35S] sulfate (6).
9. From early stage embryos, moderately abundant proteins can be detcted by immunoprecipitation, but not by simple staining procedures, such as with Coomassie blue or the more sensitive silver-staining technique. As a guideline, immunoprecipitation has been used to detect C-cadherin using two 48-cell embryos and subsequent separation by SDS-PAGE and immunoblotting using an
ECL detection system (18, and see Subheadings 3.6. and 3.7. for details of techniques). However, between 230 and 310 eight-cell mouse embryos were needed to detect E-cadherin by the same method (16). In general, the amount of material needed for this type of experiment is really a matter of trial and error. The affinity of the antibody is also a major factor with affinities of > 107/mol required to immunoprecipitate antigens efficiently.
10. A preclearing step is not essential, but usually leads to more specific immunopre-cipitation. For clean immunoprecipitates, which is especially important if radiolabeled antigens are being immunoprecipitated, an extra preclearing step is required. This is usually with nonspecific antibody coupled to protein A-sepharose for 1 h at 4°C, i.e., mouse immunoglobulins in the case of MAbs.
11. The use of a 100 mL Hamilton syringe with a very narrow bore and blunt end (point style 3) allows the liquid to be removed without any beads.
12. This can only be considered to be a very brief description, and more comprehensive reviews are available elsewhere (19). A 12.5% polyacrylamide gel is suitable for proteins between 12 and 45 kDa, whereas a 7.5% gel is suitable for 50-150 kDa proteins. Gradient gels allow the separation of most proteins of interest, and can be poured by mixing equal volumes of 5 and 15% acrylamide solutions in a gradient maker.
13. As guide to running conditions that will depend on the apparatus used: 4 h at 140 V, or 16 h at 35 V. These conditions will also vary depending on the size and percentage of the gel.
14. A more sensitive staining method is to use silver, and there are commercial kits available based on the method of Sammons et al. (20). However, staining is not usually appropriate for immunoprecipitates that invariably are associated with radiolabeling or immunoblotting detection procedures.
15. The semidry blotting technique gives a higher yield of transfer and is quicker. However, the small buffer volumes used can sometimes lead to asymmetric transfer of proteins across the gel, and it is also possible to "overblot" where the protein passes through the blotting membrane. For semidry blotting, the membrane can either be nitrocellulose or specially developed membranes, such as Immobilon P (PVDF, Millipore). In the case of PVDF membranes, they should be "activated" for a few seconds in methanol and then washed in transfer buffer, whereas nitrocellulose membranes should just be washed in transfer buffer. Wet blotting is often more effective when a PVDF membrane is used with a transfer buffer composed of 10 mM CAPS (Sigma), pH 11.0, and the transfer is carried out at 0.3 A, 60 V at 4°C overnight.
16. Colorimetric development of immunoblots probed with phosphatase and peroxi-dase-labeled antibodies is possible. However, these procedures are relatively insensitive, the color can fade, and they are generally less versatile than other techniques. Electrochemi-illuminescence (ECL) is just as easy to perform and gives a permanent record in the form of an autoradiograph, the exposure time is relatively short, and the length of exposure can be varied in order to produce the correct intensity of band. ECL reagents are readily available in kits, and therefore, the technique will not be described here.
17. Immunoprecipitation is a sensitive technique, especially when it is combined with detection by 125I-labeled antibodies. Labeling the first antibody with 125I produces a more specific reagent and, therefore, cleaner immunoblot, although it may be less sensitive, since it does not have the amplification of signal (and background), which occurs with methods incorporating another layer in the detection system.
18. The autoradiograph produced may be intensified by the use of fluorography (3H by 1000-fold, and 35S by 5- to 10-fold), and can be performed using commercially available products, such as Amplify (Amersham), or by the following method: after fixing, the gel is rinsed twice in DMSO for 45 min, and then in DMSO plus 22% (w/v) diphenyloxazole (PPO) for 45 min. The gel is finally rinsed under a stream of water for 1 h and dried before autoradiography.
19. In recent years, the general availability of phosphoimagers has replaced autoradiography and fluorography as a detection system for radiolabeled proteins. It has several advantages, including a linear response over a wide range of activities, and in conjunction with the appropriate software, can be used for easy quantitation for comparative studies.
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