Why protein sample is not able to resolve well in SDS-PAGE gel?

There are several potential reasons why some of your protein samples fail to resolve properly in SDS-PAGE despite being prepared using the same protocol. First, protein aggregation or incomplete denaturation could be responsible—this may occur due to inadequate boiling, presence of disulfide bonds not fully reduced (ensure sufficient β-mercaptoethanol or DTT), or high protein concentration leading to precipitation upon heating. Second, residual salts, detergents, or other contaminants (e.g., from lysis buffers, protease inhibitors, or incomplete clarification) may interfere with electrophoresis; consider buffer exchange or protein cleanup before loading. Third, proteolysis or sample degradation may lead to smearing—ensure immediate sample processing or inclusion of protease inhibitors and keep samples on ice. Fourth, incomplete solubilization of the pellet or uneven sample mixing may result in inconsistent loading. Finally, differences in cell lysis efficiency or localized concentration differences within lysates could explain why some samples resolve poorly while others do not; vortex thoroughly and clarify lysates by centrifugation. Running a small aliquot on a test gel or using a BCA/Bradford assay to normalize protein concentration prior to loading may help. https://www.youtube.com/watch?v=Abc9FA6p50M

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