MW of protein on SDS-PAGE different from actual mass - (Jun/02/2005 )

hi all,

recently a protein was purified from pichia pastoris in our lab. the actual mass of the protein is ~75kDa but on the gel it runs at a molecular mass of 55kDa. could anybody tell me the reason why is this the case?? the person who purified it says that the protein is the non-glycosylated form. could there be any other reason.

thanks!!

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Hi,
I am not sure if I understand your question right, but mobility of proteins on SDS-PAGE can be affected by many factors.
For example, if your protein have many hydrophobic portions, more SDS would binds to the protein molecules, and therefore the protein runs faster than actual MW.
Hope this helps.

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I think, we need to know how you got the "actual mass 75kDa", was it from a.a sequence calculation?
If the same protein you got 75kDa by, for Ex, Gelfiltration or Mass Spec, then you find it as 55kDa on SDS-PAGE, the explanation could be as mentioned above (Slab's post), otherwise, there's plenty of reasons to think of, including post-translation modification or truncated form due to proteases.

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yes, the 75 kDa is from amino acid sequence calculation. any explanation??

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I think, first you have to make sure that your protein is not a truncated form. Is there a signal peptides included in your protein, which can be processed by host cell modification machinery?

To phase out the situation as explained by Slab, I think it may worth for you to run native-PAGEs at different concentrations of acrylamides (for ex. 5%, 7.5%, 10% and 12%). You can find information somewhere regarding the calculation of MW from the native-PAGE data.

If your protein is an enzyme, and if you are sure that there is no signal peptides included in the expressed protein, the 20kDa shorter truncated form may not have activity.

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