Hello. This is the first time I am trying Protein G. I immobilized Protein G on a CM5 surface. Got about ~2500 immobilization level. Then flowed a rabbit Ab (conc. 20ug/mL) then my protein (11.5 kDa). I got a little bit of binding but got horrible drift. Any suggestions? Actually, I tried an Ab mouse also. Got an antibody response but no ag response (data not shown). I would like to use Protein G to capture other species (ie. mouse, sheep, goat, etc.) so I don't have to make a separate chip for each species. Is there anything I need to know about Protein G? Should I use Protein A? Protein A/G? Protein L? Any advice would be most helpful. But for now, the drift is my biggest problem. See sensorgram below. Thanks.
Calculate the theoretical response of your protein:
Response of protein to captured IgG: Rmax = (800 * 11500) / 150000 = 61 RU
This is the theoretical Rmax when saturating the captured antibody (per antibody 1 molecule bound). Luckily, you don’t need to saturate the ligand to get meaningful results. When I look at your sensorgram, the binding looks ok (reasonable response and curvature with this analyte concentration). In addition the reference channel 1 is decaying at the same rate so you can subtract this to isolate the interaction.
I have two publications for you which can help you further.
1. Bergstr+Âm, Gunnar and Mandenius, Carl Fredrik; Orientation and capturing of antibody affinity ligands: Applications to surface plasmon resonance biochips. Sensors and Actuators B: Chemical (158): 265-270; 2011.
2. Joss, L. et al; Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface. Anal.Biochem. (261): 203-210; 1998.
In addition you can browse
(Protein G Resins and Kits) for more information.
Also look at Tech Tip #34 which gives an overview of the Protein G binding characteristics for different IgG species.