In article <31D91BA1.41C6 at sb.fsu.edu>, Jayne Williams
<williams at sb.fsu.edu> wrote:
> Hello Everyone,
>> I am a beginner and there is probably a simple answer to my question,
> yet I have been unsuccessful in finding anything in the literature on
> this:
>> I have transfected a plasmid (pMAMneo) into CHO cells (Lec-1 line), and
> will soon induce expression of my protein (Thy-1: a cell surface
> glycoprotein, 111 aa). I have already verified the presence of the gene
> by PCR.
>> I'd like to quickly screen for protein expression by comparing SDS-PAGE
> of cell lysates (induced vs noninduced). My question: how much lysate
> (very roughly, how many cells) must I load to see my protein on the gel?
> (Of course, I have no idea at this point what level of expression I will
> get, so am only looking for a reasonable starting point.)
>> Any suggestions/references very much appreciated. (E-mail replies
> equally appreciated).
My suggestion would be, if possible, obtain an antibody against
Thy-1 and do a Western blot.
Using a quick ballpark estimate for your problem, assuming
100,000 molecules/cell, 1 million cells, and a MW of 30,000
for the product (I'd estimate that Thy-1 is around 15,000
given the number of amino acids it has, but there's no way
that I am aware of to estimate how much glycosylation has
been added to your protein):
(100,000 molecules/cell) (1 x 10^6 cells) (30,000 g/mole)
----------------------------------------------------------
(6.02 x 10^23 molecules/mole)
gives me roughly 5 ng of your target molecule. I think it's
safe to say that you won't see that against a whole cell lysate
using standard protein detection techniques. You might be
able to see your protein by isolating the plasma membranes
and only run those on your gel, which would reduce your background
protein levels at least 10-fold, but there would still be no
guarantee of identifying your protein of interest.
--
Eric Roush Phil Niekro for the HOF!
edroush at acpub.duke.edu (Net and Real!)
also coache at aol.com Send your net ballot to vinay at baseball.org
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Maddux/Glavine in 1996! It's Time for a Change-Up!
