Proteins Digest, Vol 21, Issue 9

Hi Adam,

People do often use low concentrations of detergent in ELISA buffers (0.01 -
0.1% Tween, for instance), especially in washes between incubations. It may
help cut down on non-specific backgrounds. Amounts you are likely to use
for extracting proteins are almost certainly too high though. That is,
until you dilute your sample for the assay, as I expect you might.

If you still might have too much detergent, why not dialyze your sample vs.
your assay buffer? Or if that's unacceptable, you could do a few cycles of
partial extraction buffer removal and re-dilution in assay buffer on a
centrifuge filter concentrator. Good luck, and btw do you have any affinity
column experience?

Dan


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Today's Topics:

1. Setting up an ELISA: any advice gratefully received!
(Stearns, Adam T)


----------------------------------------------------------------------

Message: 1
Date: Mon, 19 Feb 2007 15:09:20 -0500
From: "Stearns, Adam T" <[Only registered users see links. ]>
Subject: [Protein-analysis] Setting up an ELISA: any advice gratefully
received!
To: <[Only registered users see links. ].indiana.edu>
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<C35C8584C15E7D48983CF404202C411E044056C6@PHSXMB19 .partners.org>
Content-Type: text/plain; charset="iso-8859-1"

Help!

I wonder if anyone might be able to give me some advice about how to go
about
setting up an ELISA: I'm looking at fairly subtle changes in membrane
protein
expression (SGLT1 transporter) in intestinal tissue samples. I have been
doing
Western blots, and quantifying with densitometry, but I want to try to get
slightly more quantitative results. My initial main concern is the protein
extraction. Up till now, I've been using triton X in lysis buffer. Will this
interfere with the ELISA, and do I need to find a different extraction
buffer
for extracting the protein? Alternatively, does anyone know if I can remove
detergent from the samples?

With many thanks!

Adam Stearns
Boston, MA
US.





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