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Immunoblotting procedures combine the resolution of gel electrophoresis with the specificity of antibody detection. Blotting can be used to ascertain a number of important characteristics of protein antigens, including the presence and quantity of an antigen, the molecular weight of the antigen, and the efficiency of antigen extraction. This method is especially helpful when dealing with antigens that are insoluble, difficult to label, or easily degraded, and thus not amenable to procedures such as immunoprecipitation.
By taking advantage of distinct physical characteristics of different polypeptide species such as size, electrical charge, and shape, a complex mixture of proteins can be resolved chromatographically (electrophoretically) by applying the sample to a gel matrix in the presence of an electric current. A charged protein will migrate in an electric field relative to its net charge. However, as the molecule migrates through the gel matrix in response to the electric current, its mobility will be retarded as a function of the size and shape of the protein by the sieving effect of the gel matrix. Native polyacrylamide gel electrophoresis (PAGE) can be used to separate individual proteins, by size, shape and charge. PAGE can also be performed under denaturing conditions, typically in the presence of a molar excess of the ionic detergent Sodium Dodecyl Sulfate (SDS-PAGE) or under reducing and denaturing conditions (SDS-PAGE in the presence of a reducing agent such as DTT or ß-mercaptoethanol). Most often polyacrylamide gel electrophoresis is performed in the presence of SDS. Prior to resolving the sample by SDS-PAGE, the protein is denatured by heating the sample in the presence of the detergent. By disrupting non-covalent intra- and intermolecular associations, the protein is effectively rendered devoid of secondary and tertiary structure. As a consequence, the denatured protein molecules become uniformly “coated” with the negatively charged SDS at a concentration of approximately 1.2 grams SDS per gram of protein, thus giving the protein molecules a net unit negative charge per unit mass. Protein samples fractionated by denaturing SDS-PAGE are, therefore, resolved roughly according to their relative molecular weight regardless of charge (and to some degree, shape). Under non-denaturing conditions alone, however, the migration of some proteins is affected by the retention of secondary and higher order structure stabilized by covalent disulfide bonds between adjacent cysteine residues. Often, polypeptides containing intact disulfide linkages migrate anomalously by SDSPAGE. The resolution of such proteins by SDS-PAGE is influenced by their charge as well as their shape. This is due, in part, to stearic hinderence of SDS binding to the protein in regions participating in the formation of inter- or intramolecular disulfide bonds resulting in a heterogeneous charge distribution across the molecule. Additionally, the secondary structure stabilized by the disulfide linkages affects migration through the gel matrix. To alleviate this potential problem, a reducing agent such as dithiothreitol (DTT) or ß-mercaptoethanol (BME) is added to the SDS sample buffer to disrupt the disulfide bonds. Under reducing and denaturing conditions, all proteins in the sample should be resolved by SDS-PAGE according to size (molecular weight) alone. For this reason, SDS-PAGE is most commonly run under reducing conditions. A great deal can be learned about the properties of an individual protein by “running gels”. However, even more can be learned by transferring the fractionated protein sample to solid support membranes (Western blotting) for detection (probing) with specific antibodies.
The protein sample is first resolved by gel electrophoresis. Make sure that the gel acrylamide concentration is appropriate for the anticipated molecular weight of the antigen to be detected (see Appendix) and that the acrylamide solution is degassed prior to casting gel. Always pre-cast SDS-PAGE gels the day before use to insure complete polymerization for maximum resolution. Fresh ammonium persulfate and TEMED should be used to catalyze gel polymerization. Rinse wells thoroughly before applying sample to gel. Apply 10-50 :g of total cell or tissue lysates or 0.1-1.0 :g of a purified protein in 1x SDS-PAGE Sample Buffer (see Appendix) per well. If samples are to be run under non-reducing conditions, ß-mercaptoethanol and DTT should not be included in the sample buffer. Samples should be heated at 50-65°C for 10-15 minutes prior to loading gel. Samples should not be boiled as proteins containing significant stretches of hydrophobic amino acids (such as membrane proteins) tend to aggregate when boiled. It is advisable to run pre-stained molecular weight markers in one well in order to monitor the transfer of protein from the gel to solid supports during the membrane transfer step. This will also help to orient the gel during the transfer procedure. Since pre-stained molecular weight markers often do not run true to size, it is recommended that unstained molecular weight standards be run as well if an accurate determination of antigen molecular weight is desired. Following the specifications of the equipment manufacturer, electrophorese the sample through the polyacrylamide gel to resolve the protein by molecular weight. Stop electrophoresis when the Bromophenol blue dye front reaches the bottom of the gel.
Two types of molecular weight markers are available unstained and pre-stained. Unstained MW markers usually consist of a mixture of purified native or recombinant proteins of defined molecular weights. Visualizing their location on a gel or membrane requires a staining step. Pre-stained MW markers are shown in the figure “Pre-Stained Molecular Weight Marker” on this page. There are both advantages and disadvantages to using pre-stained markers. Pre-stained markers allow monitoring of protein separation in the gel during electrophoresis. They also indicate transfer efficiency in the subsequent blotting steps. However, they can be relatively expensive and the addition of dyes may affect protein mobility. Pre-stained markers may be less accurate for molecular weight determination, as dyes attached to the proteins may alter their ability to adsorb to the membrane during blotting.
The concentration of polyacrylamide in the gel can be homogenous or a gradient. The most common polyacrylamide concentration, 10%, is best suited for the separation of proteins in the range of 10-150 kDa. If unknown proteins are being analyzed or a broader range of separation is desired, gradient gels are recommended. For example, 4-12% Tris-glycine gels are suitable for proteins in the range of 30 to 200 kDa, while 10-20% gels will successfully separate proteins from 6 to 150 kDa. SDS-PAGE gels are usually 1.0 and 1.5 mm thick; however, for blotting, proteins transfer best out of thinner gels (≤1mm).
Most common gel running buffers are composed of Tris-glycine or Tris-tricine. Buffers may contain 0.1% detergent, usually SDS. Tris-glycine buffer systems are useful for separation of proteins over a wide range of molecular weights (6-200 kDa) and are compatible with denaturing or non-denaturing conditions. Tris-tricine systems are best for the separation of smaller proteins (< 10 kDa) that need to be reduced and denatured prior to loading. Both buffer systems are compatible with protein transfer to PVDF membranes. Tris-acetate buffers are sometimes used for separation of larger proteins.