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Detection of protein blots using the avidin-biotin system
The high resolution capacity of polyacrylamide gel eletrophoresis for complex protein mixtures can only be fully exploited if suitable procedures exist for the characterization of the separated components. One approach to this problem is to investigate the interactions of the separated proteins with specific antibodies or other ligands such as lectins. This can be achieved by applying antisera directly to polyacrylamide gels after electrophoresis, but this technique of "immunofixation" is inefficient and time-consuming because of the slow rate of diffusion of antibodies into the gel matrix. This has resulted in the development of methods for transferring the pattern of separated proteins out of the gels onto thin substrates. This technique is known as "Western" blotting. The most popular substrate for proteins is currently nitrocellulose. Positively charged nylon membranes have a greater protein-binding capacity than nitrocellulose and can be used successfully with antibodies, but they have not proved popular because of the lack of an easy-to-use general staining procedure.
The high resolution capacity of polyacrylamide gel eletrophoresis for complex protein mixtures can only be fully exploited if suitable procedures exist for the characterization of the separated components. One approach to this problem is to investigate the interactions of the separated proteins with specific antibodies or other ligands such as lectins. This can be achieved by applying antisera directly to polyacrylamide gels after electrophoresis, but this technique of "immunofixation" is inefficient and time-consuming because of the slow rate of diffusion of antibodies into the gel matrix. This has resulted in the development of methods for transferring the pattern of separated proteins out of the gels onto thin substrates. This technique is known as "Western" blotting. The most popular substrate for proteins is currently nitrocellulose. Positively charged nylon membranes have a greater protein-binding capacity than nitrocellulose and can be used successfully with antibodies, but they have not proved popular because of the lack of an easy-to-use general staining procedure.
Related Concept Videos
01:15Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
03:31Labeling DNA Probes
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
02:57Southern Blot
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...