Why Can Primary Antibodies Still Recognize SDS-Denatured Proteins on Nitrocellulose in Western Blot
In Western blotting, primary antibodies are sometimes able to recognize proteins on nitrocellulose membranes even after these proteins have been denatured by SDS. Several possible mechanisms may account for this phenomenon:
1. Structural Properties of Proteins After Membrane Transfer and Binding
Upon transfer to the nitrocellulose membrane, proteins bind tightly to the surface. This interaction may induce partial refolding of the proteins, allowing some secondary or tertiary structures to recover to a limited extent, which in turn can re-expose specific epitopes that are recognized by antibodies.
2. Retention of Linear Protein Structures
Primary antibodies are typically developed to target specific linear epitopes, such as defined amino acid sequences within the protein. Despite SDS-induced denaturation, the linear arrangement of amino acids often remains intact, enabling the primary antibody to bind to its specific epitope on the target protein.
3. Antibody Specificity and Type
The ability of an antibody to recognize denatured proteins depends largely on its type. Some antibodies are raised against the native conformational structure of the protein (referred to as “conformational antibodies”) and may fail to recognize proteins that have been denatured by SDS. In contrast, another class known as “linear antibodies” is generated against continuous peptide sequences and retains the capacity to recognize denatured proteins, including those processed through SDS-PAGE.
4. Effect of Blocking and Incubation Steps
During the Western blot procedure, non-specific binding sites on the membrane are typically blocked using agents such as non-fat milk or bovine serum albumin. This blocking step enhances the specificity of antibody binding by reducing background noise, thereby facilitating the detection of target proteins.
Therefore, although proteins are denatured during SDS-PAGE, linear epitopes often remain accessible and can still be specifically recognized by primary antibodies. This underlies the effectiveness of antibody-based detection of target proteins in Western blot experiments.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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