Do proteins break up on SDS PAGE treatment?

Ian A. York iayork at panix.com
Fri Nov 17 12:29:44 EST 2000


In article <8v3lrs$7j6$1 at oyez.ccc.nottingham.ac.uk>,
Student <mucineer at iname.com> wrote:
>Do proteins break up by heating with SDS buffer at 70 degrees for 10
>minutes? I found that when I ran a non-reduced SDS PAGE gel on a purified
>protein (IgG purified from a protein column), although the vast majority of
>the product is at the right size, there are some faint bands scattered
>below. I am quite sure that they are not contaminants because when I ran a
>reduced gel, they weren't there (just one very faint band as opposed to 4 or
>5 in the non-reduced gel), yet they both came from the same sample!

If you did not see the bands in the reduced gel, but did see them in the
non-reduced, it seems unlikely that it's a question of degradation or
cleavage; the reducing gel should show that more clearly, not less.

Possibly your protein has internal disulphide bonds (perhaps only in a
small fraction of the total; perhaps artifactually introduced, or
misfolded during synthesis); if so, the non-reducing gel will allow these
proteins to remain in their more compact form (folded, rather than
completely linearized) and so they'll run faster than the completely
linear molecule.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England






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