Hello!!
I am concentrating cellular supernatants to detect TGFb by ELISA. I obtain a poor signal, thus I concentrate the supernatant using Amicon Ultra-2 Filter Devices or Vivaspin 2 centrifugal concentrators.
When I concentrate the supernatant 10 times, I only get twice the signal in the ELISA. Is this normal, or should I expect an ELISA signal 10 times greater?
Thank you so much in advance!!
Dear Claudia,
First off, no. 10fold more signal would mean 100 % recovery, and that is not going to happen. However, the 20 % you are seeing seems quite low. When using ultrafiltration I usually got 30-80 percent, depending on the protein I was looking for (membrane proteins are usually the worst). Try washing your membrane, rinsing it with the retentate. May release some protein that's stuck to the membrane. Also you might want to reconsider type or size of membrane that you are using. Your molecular weight cut-off should be a safe distance - maybe half to a quarter - smaller than your proteins molecular weight. Also be aware of di- and multimer formation, as there's usually no solubilizing or denaturing substances in your culture supernatant. Hope that helps, atb Christoph
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