Slowly migrating EGF receptor in SDS-PAGE

[John A. Newitt]

In article <3l4q10$cj1 at abc.ksu.ksu.edu>, maraman at ksu.ksu.edu (Dayanidhi
Raman) wrote:

> Hi, I immunoadsorbed the EGF receptor and eluted it, dialyzed to remove
the     detergent in the presence of added lipid, assayed for tyrosine
kinase activity. The prep. was run on 7.5% SDS-PAGE after heating the
sample at 60C for 3 min.   in Laemmli's sample buffer. The autoradiogram
has all receptors at the very top of the gel. Need help desperately!.
> A

You don't mention whether your sample buffer had reducing agent present. 
For a reducing gel, you should include 100 mM DTT (or 2-mercaptoethanol,
but less effective) in your sample buffer.  Also, increase your heating
step to 30 min at 60-65 degrees.  Make sure there is enough SDS in your
sample buffer for the amount of protein you are loading.

John A. Newitt  <newitt at ncifcrf.gov>
National Institutes of Health
Bethesda, Maryland  USA