Purifying Protein Samples to Remove or Reduce SDS? - Need to remove or reduce SDS in protein extract so ELISA will work! (Apr/30/2010 )
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!
Have you tried potassium salt precipitation of SDS?
One suggestion that can be quickly tried out....SDS precipitates out in the cold right? Maybe you can try snapfreezing your protein samples and thawing on ice.One freeze thaw cycle doesnot affect our protein activities much, but I guess it may vary from case to case...also I don't know if SDS precipitation in this manner will pull down some of your protein with it... you may want to give it a try with a small batch of protein. Commonly used methods for removing SDS followed in our neighbour lab is to either pass the sample through a Desalting coloumn or use the commercially available Pierce buffer that precipitates out detergents.....
you can dialyze against triton x-100 to remove sds from the protein (drop dialysis if your volumes are very low).