Did you use b-mercaptoethanol or DTT in your Laemmli buffer and did you boil your samples ? The mobilities of proteins depend on reduced or non-reduced conditions (if they have disulphide bonds) and on the boiling or not.

1. Probably your running is very rapid fro 6% gel?

2. Both BSA and HSA could form relatively stable dimers that appear to correspond twice of lower mol mass.

3. You description suggests that there is a problem with gel or running. Check SDS concentration and use beta-mercaptoethanol.

Thanks for the answers. I used DTT and boiled the samples in laemli buffer for 5 minutes. I also thought it might be the reducing conditions that may affect it but why would these proteins run at different molecular weights and other recombinant proteins show up at the correct molecular weight under these conditions?

Thanks.

Natasha,

I've just looked your gel (not noticed yesterday). We never observed such a difference of 20 kDa. I think that protein was incompletely denatured and therefore, no relation is observed between protein conformation and mol. mass of markers. The problem most probably is caused by preparation of samples and/or running conditions. Check both sticking and running gels, change buffer, heat samples in beta-mercaptoethanol at >90°C, etc... and repeat SDS-PAGE. I encourage you, It should work!

VS

Thanks so much to Prof Sakanyan from France. You really saved my neck. Your suggestions worked out well. My gel pic is attached and shows the HSA and BSA in the correct molecular weight. Thanks.

Dear Natasha,

Bravo! Further success to your research.

Vehary