Most likely, you are witnessing cross-linking of monomers due to the formation of spurious disulfide bridges. This occurs when the sample buffer has lost its reducing power due to oxidation of the reducing agent (mercaptoethanol, DTT or trimethylphosphine). Mercaptoethanol is less prone to the problem because it is added at a much higher concentration than the two other reagents.

Dear Divja

is it your protein a membrane protein? because sometimes membrane proteins may run aggregate in sds expecially if you are boiling the samples before loading.

Divya Tarwadi , does the protein have cysteine residues?

Also, the point raised by Manuele Martinelli is important. Try incubating the samples for 20 min. at 37*C instead of the standard 5 min. at 98*C.

Pierre Béguin sir, to increase reducing power of sample buffer i have added more mercaptoethanol in my working dye(1:30as per guideline) but i didnot able to load my sample in well ,it was spling out this happend only with dye having more mercaptoethenol concentration.

Manuele Martinelli yes, my protein in membrane protein and i boil my sample with dye. i run gel many times still it is showing same band size.

Alejandro Martin thank you so much for your suggestion.

Dear Divya

if your protein is a membrane protein try to run again the gel folliwing the suggestions about sample preparation of Alejandro Martin.

however if it will nof succeed don't worry membranr protein are diffixult to manopulate and it is possible that show atrange behaviours in sds page but i think that if you are sure that it is your protein because you purified it with an affiinity tag or recognize it with an antibody in wb or by mass specttrometry analisys of sds-page band , its more important to understand which is it aggregation state in non denaturing conditions to chhose the best detergent and buffer.

good luck

Manuele

Manuele Martinelli thank you so much