Ian A. York
iayork at panix.com
Mon Sep 13 07:31:12 EST 1999
In article <37dcd34f.5502935 at news.univie.ac.at>,
Martin Offterdinger <a8803349.nospam at unet.univie.ac.at> wrote:
>>I'm doing IPs with 20 ul of a 50% protein A-Sepahrose in a total volume
>>of 1 ml of lysis buffer... I find that I can barely see a 'pellet'
>>forming after centrifugation. Am I using too little protein
>1) A sepharose pellet is rather clear and not easily visible
>2) Are you sure that you added any beads? This is not too trivial as
>the beads are difficult to pipet as a suspension. I usually do the
>following: Suspend beads vigoruosly
>Cut a yellow tip with a razor blade. Use this broadened tip to pipett
All good points, but 20 ul of a 50:50 protein A solution is going to give
a very small pellet, period.
Whether it's "not enough" depends a little. It's probably plenty to
adsorb all the Ig in your immunoprecip. The concern would be that if you
lose a little bit of the pellet you might actually be losing a sigificant
amount of your protein of interest--e.g. if 1 ul sticks to the outside of
the pipette during transfer a couple of times, you're down to 80% of your
actual start, which could be a problem if you're quantitative. It's also
harder to work with a small pellet in general, especially if you're not
used to them.
I generally use 30-40 ul of a 50% prep. There are two minor
disadvantages: it costs twice as much, and if you want to load a very
small volume on your gel it might be a little trickier. Neither, in my
humble opinion, is a big deal.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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