multimer on denatured SDS-PAGE?

Stephen C. Dahl stebby at welchlink.welch.jhu.edu
Tue May 5 13:53:35 EST 1998


Cornelius Krasel (krasel at wpxx02.toxi.uni-wuerzburg.de) wrote:
: Rick00100 <rick00100 at aol.com> wrote:
: > I'm a newbie in protein work and Western, and recently I'm trying to raise
: > antibodies to my protein, which is about 22kd.  I got some polyclonal Ab,
: > and when I do a Western with CHO cells-transfected protein, I would not see
: > a band with one of my Ab.  However, after deglycosylating the cell lysates,
: > I can see a band at around 40kd, and a faint band at 22kd.

: It's possible that the 40 kDa band is a dimer. Since your protein is
: a membrane protein (why else would you want to deglycosylate it), this
: is not entirely surprising. Sometimes it helps to omit the boiling step
: (at least this is true for some G protein-coupled receptors).


Agreed.  We work on proteins with multiple membrane spanning domains.  One
of them becomes a mass of goo that will not enter the gel if its boiled.
Heating at temperatures no greater than 65' for 3--5 minutes does the
trick. 

Hey, and Rick, if you're here at JHMI how come you're not on welchlink?



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