Problems with RIPA in ELISA - (Aug/02/2010 )

We are using RIPA buffer to extract protein from a mammalian tissue. The problem we encountered is that the RIPA buffer seems like it interrupted the reaction in the ELISA even with the 1:2 dilution of RIPA buffer. We have been considering using PBS as a protein extraction buffer but we are worried that PBS will not be sufficient to extract the protein without detergent. Any suggestions would be greatly appreciated.

dialyze the extract against a more compatible buffer.

mdfenko on Mon Aug 2 14:36:30 2010 said:

Thank you so much for your reply mdfenko. We actually don't have any experience in dialyzing an extract. Do you have any recommendations or could you point us towards a post with instructions? Also, would you have any suggestions for a more compatible extraction buffer? We are trying to measure the levels of IL-6 from mammalian tissue.

Rdub on Mon Aug 2 14:46:37 2010 said:

here are a couple of documents from thermo-pierce. they describe dialysis and available (from them) devices. you select the device based on the volume you want to dialyze.

if the volume is small enough then you can perform "drop dialysis". float a dialysis membrane (preferably one layer, which can be cut from tubing) on a beaker of buffer and place a drop of your solution on it.

as for buffers, ripa should be okay.