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Purification of Protein Extract for ELISA

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#1 WolfeMD



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Posted 30 April 2010 - 06:13 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 questions are thus:

1. Is there a level of SDS at which ELISA won't be inhibited?
2. Will dilution to that level kill my accuracy? (Maybe that is for me to determine experimentally)
3. Is there a method for getting the SDS out of my sample that is affordable and relatively high-throughput?

Thanks for all input!


#2 sgt4boston



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Posted 30 April 2010 - 06:57 AM

If the surfactant works well to extract your protein of interest and that protein is relatively stable in that buffer/matrix for a reasonable period I would not change that part of your method.

Remove the surfactant in the samples by dialysis or other method. I believe Thermo Fisher/Pierce has devices methods for rapid dialysis OR Millipore now Merck has devices/methods as well. Both of these are relatively inexpensive or use common dialysis tubing.

Now you will be able to keep your extraction method intact, remove the surfactant, and not have to worry about any dilution (dialysis may slightly change the final volume of sample). Be sure to measure starting volumes and concentrations pre and post dialysis.

Good luck!

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