quantify protein on beads? - (May/31/2005 )
Hi, I'm using GST fused protein in my enzyme activity assay. I have to use 50% slurry to keep the enzyme active. But I don't know how to quantify the protein on beads. I tried to elute the protein and quantify the elution, but then I found out there were still a big amount of protein on the beads. Does anyboday know if there is a way to quantify the protein while it's on the beads? Or is there a way to take the protein off the beads more completely? Thanks a million!
did anybody do this before? thank!
I think you can use SDS-PAGE method to test the slurry of your beads. When you add SDS loading buffer to the slurry and heat it, the protein will be denatured and can be seen on your SDS gel as you do your normal SDS gel for proteins.
Note you have to figure out the control and calibration when you quantify the protein on beads. Good luck!
Thanks! I also tried boiling the beads (there is a paper used this method), but without the SDS-PAGE loading buffer, very little protein came off . I will try the one you said.
I agree with the reply from the other person. I also found out that the beads remain on the top of the gel. the beads are too big to go throght the gel.
hope it work!!