Concentrating dilute protein samples for use in elisa - (Jan/23/2008 )

Hi there
I am new to protein work and hope to do an elisa with some heart lysates I have. My problem is that the lysing buffer I have used seems to cause a colour change in the BCA assay so that I can't determine the concentration of the protein and also I suspect my samples may be too dilute- I need to concentrate them. I have tried both TCA and acetone precipitation but find it impossible to resuspend the pellet in a suitable buffer (I'm using PBS). I suspect the proteins are denatured and are forming insoluble aggregates?

Can anyone suggest a method for concentrating proteins that will cause minimum denaturation, a method that is compatible with elisa? Can anyone recommend a good buffer exchange kit?

Thank you!

Alison

U can use small concentrators with appropriate pore sizes.
u can find good information in company web site.

i would not do precipitation of samples if ur down stream application is ELISA.

regarding protein quantification, try bradford, if does not work, go for 280nm....

gud luk

Thanks for your reply.
Alison