Can WB Without SDS Work for Small Proteins (10KD)? Is Tris Used Instead for Gel Balance
Small proteins (10 kDa) are typically analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in Western Blot (WB) experiments. SDS, as a surfactant, denatures proteins and imparts a uniform negative charge, enabling their separation based solely on molecular weight.
In the absence of SDS during electrophoresis, proteins are not fully denatured. As a result, their migration behavior is influenced not only by molecular size but also by their native conformation and intrinsic charge, leading to size-independent mobility. Under such conditions, non-denaturing (native) electrophoresis is generally applied, where proteins preserve their native structure and charge characteristics.
When SDS is omitted, buffer systems such as Tris-glycine or Tris-acetate are commonly employed to maintain pH; however, these buffers do not induce denaturation or confer uniform charge to proteins. Therefore, if the experimental goal is to separate proteins based on size, the exclusion of SDS is not recommended. Conversely, if the aim is to preserve the native structure and biological activity of the protein, non-denaturing electrophoresis conditions may be considered.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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