When I run my protein lysate in 10% sds-page gel, while loading protein samples it coagulates and does not resolves properly in gel giving a smeared ponceau stain as shown in the photo attached. But it is not happening with all samples but few. even when all the samples are made together using similar procedure. what could be the reason for same ? some samples made with same procedure during same time are resolving fine while some are not.
What is the running buffer and sample buffer composition, and the load amount of the protein?
Too much protein loading,
diluted SDS or an incorrect recipe of Laemmli buffer,
Some membrane protein solubility issues,
Some of the main reasons for coagulation, probably encountered during sample introduction to 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.
Do you mean by "coagulates" that the sample precipitates in the gel pocket after loading? Do you have potassium ions in your sample (or guanidine) which could lead to insoluble K-SDS upon cooling down but soluble in the heated sample. Other than that your protein lanes do not look that bad, I've seen way worse protein gels. The four samples on the right hand (or upper) side of the lower gel seem slightly overloaded and/or contain a bit more DNA which could lead to the smear, but again, this is not a problem. If you have a whole cell lysate you can't expect many dicrete bands, there will be a lot of stained protein throughout the whole lane with some more discrete bands, as is the case in your gel.
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