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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 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!
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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.

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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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.

Cis or trans? That's what matters.
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