strange c-terminal deletion casue protein run high in SDS-PAGE

D.K. dk at no.email.thankstospam.net
Tue Oct 2 08:03:19 EST 2001


maildie at yahoo.com (andor) wrote:
>Many thanks,A.G.E
>I did run a mixture on SDS-page and get the strange results I
>reported.
>The sample (boiled in a standard loading buffer for 10 minutes)I
>repeated 3 times and am sure those strange "HIGH" result is a real
>one.
>Some guys in our lab suggest that post-translation modification may
>sometimes change the status that SDS binds my protein, so the band may
>run higher or lower a bit.
>If the last amino acid I deleted carried an unknown factor without
>which protein shows a higher band ,how come "Del-2" run higher than
>"Del-1"(Both of them lack the last amino acid and only 1 Aa
>difference)? given that one amino acid difference could not be
>detected on 15% SDS-PAGE.

You keep repeating this, but it is not always true. In fact, even lesser 
changes in much bigger proteins are sometimes detectable. A classic
example is phosphorylation-induced shift - a single phosphate group
in 100 kDa protein can cause it run slower ("higher") in the gel. 

What you detect on SDS PAGE is number of SDS molecules bound 
to protein. This varies depending on protein composition and structure 
(yes, structure because SDS does not denature all proteins 100%). 
Are you, by any chance, deleting Asp and Glu? 

DK




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