In Western blotting, why can the primary antibody still recognize proteins on the nitrocellulose membrane that have been denatured by SDS? What does the primary antibody recognize?
In Western blotting, if proteins on the nitrocellulose membrane have been denatured by SDS, but the primary antibody can still recognize them, possible reasons are:
1. Protein structure after transfer and binding to the membrane:
When proteins are transferred to the nitrocellulose membrane, they bind tightly to the membrane. This binding may cause partial restoration of the secondary or tertiary structure of some proteins, thereby re-exposing certain epitopes.
2. Linear structure of the protein:
Primary antibodies for proteins are usually designed to target specific linear structures (e.g., specific amino acid sequences). Even when proteins are denatured by SDS, their linear structure remains, allowing the primary antibody to bind to specific regions of the target protein.
3. Type of antibodies:
Firstly, the type of antibody determines its ability to recognize denatured proteins. Some antibodies are prepared against the native or conformational structure of proteins (commonly known as "conformational antibodies"), and these may not recognize proteins denatured by SDS. However, another class of antibodies is prepared against linear or continuous peptide segments of proteins (known as "linear antibodies"), and these can recognize denatured proteins.
4. Blocking and incubation process:
In Western blot experiments, non-specific sites on the membrane are usually blocked by blocking agents (such as non-fat milk or bovine serum albumin), which can also help improve the specificity of antibody binding.
Therefore, even if proteins are denatured in SDS-PAGE, certain epitopes in the linear structure can still be recognized by specific antibodies, which is why we can still detect target proteins with antibodies in Western blot experiments.
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