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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!



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

-donot lie for ever-

Thanks for your reply.