question about protein molecular weight - (Dec/10/2007 )

Now I have a question about my protein sample. This protein has 3 Cys. It is about 11.8 kDa. I did purify this protein this August and sent to do Mass Spectrum. It showed a good peak at 11.8 kDa. However, I redid the expression and purification this time. I ran on column 3 times: 1st, affinity column; 2nd, affinity column (after digestion with thrombin); 3rd, gel filtration column. I sent to do MS again after buffer exchange with pH 4 dd water. However, this time, there are 3 peaks showing up. One is 11.8 kDa. The other two is 12.1 kDa and 12.4 kDa. Each one is 266 Da more than the previous one. I did gel electrophoresis on mini-gel, there is only one band after coomassie blue staining. I am quite curious about what happened. I guess that the protein has been modified with some kind of fatty acid. Can you help me with this question? Thank you.

you probably got a thick band in your sds-page. the three proteins are too close to separate on a minigel with enough protein to stain with coomassie. if you want to see separation of the three, load a lot less and stain with silver. you may see all three bands.

if you have free cysteines then you may have something binding through the sulfhydryl group. this bond will be cleavable with dtt or 2me. you can check on sds-page with reducing sample buffer.

i suddenly realized that when i did electrophoresis, i added beta-ME to the running buffer. so even the cys on the protein is binding to other groups, it will be reduced. therefore, even the 'changed' protein will be shown up the same as the native protein. is that a possible reason that there is only one band on the gel? thank you.

That might be the case, but the resolution power of SDS-PAGE may not be enough to separate these, if these are not charged groups. If you dont use 2ME, you may get multiple bands due to self-conjugation. So you are in a tricky position. But isnt that fun when you finally figure it out one day?

thank you. if i run the native gel electrophoresis, would i be able to tell the difference between the native protein and 'changed protein'. i assume that the attached group is of not charge, so the changed protein will be able to show up in the gel?

You may want to test experimentally with 10% discontious native PAGE, with and without the presence of 2-ME.

I did the protein gel electrophoresis on continuous native PAGE without 2-ME. And I noticed that there are two bands on the gel.
What happened? Is one of them the monomer, and the other dimer because they form intermolecular disulfide bond? Thank you.

Most likely, but only when you have done 2-ME Native PAGE and see one band, then you can say for sure.

But i have already done SDS-PAGE with 2-ME. They just show up as one band.
Is it still necessary to do native gel with 2-ME? I think they are in the same mechanism.