Protein is running little higher than expected on SDS-PAGE gel

[rbrgouda]

Hi,

I have expressed several recombinant proteins in E.coli.

But, I never encountered a strange problem like this before.

Currently two of the proteins I'm trying to express in E.coli are running about 4-5 kDa bigger than expected size. If it had happened with only one protein then I would assume that particular protein property makes it run it little higher. At this point of time I'm thinking of changing the protein sample buffer containing higher percent b-ME and SDS.

Please help me to overcome this problem.

 

Rbrgouda:confused:

[CharonY]

a) what markers do you use

b) are they prelabeled?

c) are the proteins in question membrane proteins?

[rbrgouda]

Hi Charon,

a) I use SDS-PAGE low range molecular weight standards (Bio-Rad)

b) They are not prelabeled

c) Proteins are not membrane proteins, they are helicase and single stranded DNA binding (SSB) proteins.

[CharonY]

OK, based on that the only question is whether your old proteins still run as they are supposed to. If they do then it is obviously a property of your proteins.

IIRC helicases had a different electrophoretic mobility as compared to their size, but I haven't got any calculations at hand. Deviations of up to 5kD from any given markers are, as a whole, not that unusual, though.

The important bit is to establish the identity of the protein in question. If you have access to an MS I would try that instead of trying to tweak the condition to have it match the size.

[rbrgouda]

Hi Charon,

Thanks for the reply. Your information helped me to understand more about my proteins. As you said, it should be the property of the protein itself. Both the protein show deviation less than 5 kDa. I did MS to confirm the presence of both the proteins. I didn't try to estimate the mass of the full size protein, instead I was looking for the presence of these protein peptides after trypsin digestion. If it is possible in MS I will estimate the mass of the full length protein.

Thanks

Rbrgouda

[CharonY]

Both are useful (i.e. petpide mass fingerprinting and full sized MS). The first is more convenient, however there is always the possibility that somehow your recombinant product got something that it should not have. You would have to check all acquired spectra, although unidentified peaks could also have other sources). Full sized MS (e.g. with a MALDI) can give you at least the average mass of the thing. You would have to use the right matrix, of course.