Can gel filtration effect buffer composition? - (May/05/2008 )
I am currently trying to purify a solubilized receptor using gel filtration. To test for the presence of my solubilized receptor in solution i apply it to a receptor binding assay (RBA) and when it is present it reduces binding of my fusion protein ( heat insensitive AP fusion protein) to my membrane, which i pellett and test using p-npp (an ap detection substrate, amount of AP determined using a spectrometer, more absorbance = more fusion protein bound). I compare normal binding of the fusion protein to binding when a solubilized receptor is present. Binding is decreased (absorbance less) when solubilized receptor is present.
Interestingly after gel filtration and concentration of my sample, when I apply fractions obtained from the column to my RBA (100 ul into 500ul) I get greater then normal binding (higher absorbance). I dont understand this as my column buffer is the same as the buffer i use to solubilize my membrane in (HBSS) and when i add HBSS alone there is no effect to binding. Could filtration of my buffer in a 45 micron filter or sterilization of the column buffer alter its actions in my RBA? Or is the filtration column altering my buffer composotion? Any help would be appriciated.
J
Interestingly after gel filtration and concentration of my sample, when I apply fractions obtained from the column to my RBA (100 ul into 500ul) I get greater then normal binding (higher absorbance). I dont understand this as my column buffer is the same as the buffer i use to solubilize my membrane in (HBSS) and when i add HBSS alone there is no effect to binding. Could filtration of my buffer in a 45 micron filter or sterilization of the column buffer alter its actions in my RBA? Or is the filtration column altering my buffer composotion? Any help would be appriciated.
J
Off the top of my head, I'd say you might have removed some inhibitors.
Did you have the same detergent concentration in samples and elution buffers?
Swanny is probably right.
yes i think that is the problem. I have done a trial run "ballparking" the detergent concentration of my previous sample, and my results improved, it also increased the liability of the receptor. I will be sure to make the detergent concentration in the elution buffer the same as the concentration in the samples. THANKS!
J