protein help needed
J C Ford
jford at grove.iup.edu
Mon Jan 6 17:30:35 EST 1997
>> In <32CED7DA.3ED6 at jcu.edu.au> lachlan harris
>> <lachlan.harris at jcu.edu.au> writes:
>> >Can anyone help me with this problem? I have a pure microbial protein
>> >which when run on an SDS non-reduced (ie no mercaptoethanol) PAGE gel
>> >runs at approximately 50 KDa. When the same protein is run under
>> >SDS-reduced conditions two subunits appear- one running at approx 40
>> >and the other at approx 60 KDa.
>> >It was my understanding that the two subunits should add up to be the
>> >same size as the protein run under SDS-non reduced conditons. But this
>> >isn't happening. Does anyone know why?
You've got to remember that a protein runs according to size, shape, and
charge, not molecular weight. When you run the unreduced protein, it has an
APPARENT molecular weight of 50 kDa. When you reduce it, it gives two bands,
one at 40 kDa, the other at 60 kDa. It seems to me most likely that the 50
kDa estimate is wrong - the protein is likely running fast - either it's quite
compact, highly charged, or in some other way different from your standards.
It's certainly - unless you messed up - disulfide-linked into a compact form
in the absence of BME.
Check the amino acid composition - is the protein likely to be highly charged?
More information about the Methods