Proteins are visualized with a variety of dyes.
treatment and the resulting peptides are analyzed by mass spectrometry (see below). This allows unambiguous identification of each protein spot.
To detect the presence or absence of a particular protein, a sensitive silver stain is used. For quantitation of protein spots either Coomassie blue or fluorescent dyes are used to stain the gel. The gel is then scanned with a laser. A variety of fluorescent dyes are used, especially in large-scale proteomics work. SYPRO Orange and Red dyes bind proteins non-covalently. These are non-fluorescent in aqueous solution but fluoresce when in a non-polar environment, including when bound to protein-SDS complexes. It is also possible to covalently label two protein samples with differently colored dyes, say methyl-Cy5 and propyl-Cy3, and run them on the same gel. Differences between the samples may then be directly visualized (pure red and green spots indicate that a protein was found in one or other sample and a yellow spot indicates its presence in both).
Proteins may be detected by binding to a specific antibody.
Much like a Southern or Northern blot identifies one specific DNA or RNA (see Ch. 21), a Western blot allows detection of a single protein within a sample of many proteins. First, the sample of proteins is separated by size using SDS-PAGE or 2D-SDS-PAGE. The proteins are then electrophoretically transferred to a solid membrane such as nitrocellulose. Electrophoresis moves the proteins from the gel and onto the nitrocellulose where the proteins adhere (Fig. 26.05).
To detect a specific protein, an antibody to that protein must be available. An antibody can either be produced for the protein of interest or sometimes purchased commercially. The nitrocellulose membrane itself has many non-specific sites that can bind proteins, including antibodies. These sites must be blocked with a non-specific protein solution such as re-hydrated powdered milk.The primary antibody is added in the milk solution and binds to the protein of interest. The antibody protein complex is detected using a secondary antibody that has a label attached to it (Fig. 26.06). Often a reporter enzyme such as alkaline phosphatase is linked to the secondary antibody, and the addition of lumiphos or X-phos to the blot allows detection of the protein band (see Ch. 21).
Automated mass spectrometry may be used to identify proteins in large numbers of samples.
Analysis of proteins and peptides by mass spectrometry relies on several recently developed techniques that are both extremely accurate and may be automated. The two most important are matrix-assisted laser desorption-ionization (MALDI) and electrospray ionization (ESI). Mass spectrometry (MS) measures the mass to charge ratio (m/z) of ions and allows derivation of the molecular weight. Before MALDI and ESI large heat-labile molecules such as proteins could not be analyzed by mass spectrometry.
In MALDI, gas-phase ions are generated from a solid sample by a pulsed laser. First, the sample protein or peptide is crystallized along with a matrix that absorbs at the wavelength of the laser. Matrix materials are usually aromatic acids such as 4-methoxy cinnamic acid. The laser excites the matrix material, which transfers the energy to the crystallized protein. The energy then releases ions, the size and charge of which are unique to each protein. The ions are accelerated by a high voltage electric field and travel in a vacuum through a tube to a detector. The time-of-flight (TOF)
electrospray ionization (ESI) Type of mass spectrometry in which gas-phase ions are generated from ions in solution
MALDI Matrix-assisted laser desorption-ionization. Type of mass spectrometry in which gas-phase ions are generated from a solid sample by a pulsed laser mass spectrometry Technique for measuring the mass of molecular ions derived from volatilized molecules matrix-assisted laser desorption-ionization (MALDI) Type of mass spectrometry in which gas-phase ions are generated from a solid sample by a pulsed laser time-of-flight (TOF) Type of mass spectrometry detector that measures the time for an ion to fly from the ion source to the detector Western blot Detection method in which an antibody is used to identify a specific protein
FIGURE 26.05 Electrophoretic Transfer of Proteins from Gel onto Nitrocellulose
A "sandwich" is assembled to keep the gel in close contact with a nitrocellulose membrane while in a large tank of buffer. The sandwich consists of the gel (gray) and nitrocellulose (green) between layers of thick paper and a sponge (yellow). The entire stack is squeezed between two solid supports so that none of the layers can move. The "sandwich" is transferred to a large tank filled with buffer to conduct the current. As in SDS-PAGE, the proteins are repelled by the negatively charged cathode and attracted to the positively charged anode. As the protein move out of the gel, they travel into the nitrocellulose where they adhere.
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