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2ME and SDS

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2ME and SDS

Postby biology_06er » Tue Nov 15, 2011 8:56 am

Hi there,

So I've recently been tryed to purify my protein and everytime I ran a SDS gel, I had a band at 120kda and my protein is around 30kda. I tried size exlcuion, ion exchange, but that 120 band was allways there...so then it was found out I didn't add 2ME to my reducing buffer :S...and once it was added, viola, the 120 band was gone. So my question is, if 2ME's role is to break the disulfide bonds to make a monomeric protein. So in the case the bonds arent broken, the size is 4x--does this mean the protein is made up of 4 subunits?...i don't really get the fact the a computer program says the estimated MW is 32kda but then this gel says its 120 if bonds arent broken--does it mean my protein is able to form a tetramer but in nature the protein is actually only a monomer?

Hope I make sense!
Thanks
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Postby canalon » Tue Nov 15, 2011 10:44 am

As far as I can see your problem, it tells you that the computer is unable to guess that the protein will form a tetramer in nature. Hardly surprising that.... The computer will just the size of the sequence you fed it and calculate a size based on that. It does not magically analyse the protein to infer disulfide bonds and such.
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Postby JackBean » Tue Nov 15, 2011 11:25 am

As canalon said, the program calculates MW of only one subunit, but obviously, they are connected by disulfide bonds to form tetramer.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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