Dear All,
My protein has a heparin binding site,and I am planning to pull down the protein complex to see whether my target protein is binding to that protein or not.I have one heparin agarose saline solution type I beads.How much beads you think is enough to detect the presence of my target protein in WB. I checked the binding affinity of my protein and it is very high. it will give you a sharp peak even with 250 nM concentration. Binding capacity of the beads is almost 20 mg/ml . I used 100 ul beads which seems to be quite high for me. Anyone has used that beads before for an IP/Pull down? I should also mention that 600 ug of total whole cell lysate(WCL) was used. As my membrane associated protein is quite tough to get in supernatant,I hads to use WCL .
Dear Abir, I think it is a good idea to determine how abundant your protein of interest in the WCL before you proceed with the affinity pulldown experiment. A simple WB would give you a rough idea of its abundance. The point is that you don't want to use too much beads relative to the protein of your interest for the pulldown. If the binding capacity is 20mg/ml of beads, then 100µl would have the capacity for 2mg of your protein. I think you probably want to use a ratio of beads to your protein of interest of 1:1 or less. If you use too much beads, you will likely have high background non-specific pulldown, and a minor point is that you would unnecessarily waste your beads. Good luck with your experiments.
Hi @hua xiao, thanks for your comment. My protein expresses nicely. Wb detected very early during visualization.
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