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Help with protein extraction buffer problem!?


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#1 WolfeMD

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Posted 30 April 2010 - 06:41 AM

Hi all,

I've been developing a sandwich ELISA for a ~100 kDa HSP from plant tissue. The ELISA works in principal, the problem is protein extraction.

When we used Western Blots we extracted in a 0.4% SDS-based buffer. This kills the antibodies in my ELISA and diluting the buffer down to as low as 0.125X (0.05% SDS) didn't work either.

I've been trying to use Triton X-100 as a replacement surfactant, but in terms of squeezing the proteins out of the tissue, Triton is whimpy and I can't trust it to get a full sampling of the tissue protein present in my samples.

So my question for this forum is whether someone could point me in the right direction of methods for purifying an SDS-laden protein sample such that the protein concentration is not too greatly reduced but the SDS is essentially removed, perhaps exchanged for a different detergent?

I will say that dialysis is probably not a good option. I need this to be high throughput, affordable for many (hundreds) of samples, and work on volumes of 1.5 mL or less! Unless there is some form of small volume microcentrifuge tube-based dialysis?

Thanks for all input!

~M

#2 Gerard

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Posted 01 May 2010 - 04:39 AM

A pierce kit fos SDS removal.
http://www.piercenet...fldID=02050702#

I found also this link: http://www.scientistsolutions.com/t7420-sds+removal+through+dyalisis.html

Edited by Gerard, 01 May 2010 - 04:40 AM.

Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".




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