SDS-PAGE protein processing

Peter Cherepanov peter.cherepanov at med.kuleuven.ac.be
Thu Oct 31 04:24:08 EST 2002


Can it be that sample preparation was different? For example, a human brain
sample is, I guess, not an easy one to find. Were all your samples boiled
before PAGE? It is sometimes important: for example, one protein I worked
with (intracellular protein) migrated much higher in the gel (3xMW) unless
you boil it before (incomplete denaturation, I think), even though, the
control HeLa cell lysate that came with the antibody I was not supposed to
heat, according to the company protocol. Another thing is salt
concentration, but that you would notice during the electrophoresis, of
course.

Sample concentration is very important too. Chemiluminscent detection is not
very reliable sometimes. If a sample is more concentrated you will see
unsecific bands that are not present in the other preps. May be the higher
band you see is not relevant at all.

other "theoretical" things I can think about:
post-translational modification (Phosphorylation shifts your band up, for
example. Ubiquitinylation?).
alternative splicing
degradation of this protein in some samples  ....

all then depends on your sample preparation (Protease, phosphotase, ...
inhibitors; etc).

good luck,

Peter

You wrote:

>2 different samples: term placenta and cultured term cells. Using one
antibody with two controls: rat brain and human brain.
>I get a band at expected mwt in term placenta and rat brain but then when I
use human brain and cultured term cells I get a >matching higher mwt band in
both the human brain control and cultured cells.
>All of these bands disappear with preabsorption!
>I hope this is clearer.



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