Forum :: capture immobilization levels on a antiHis antibody


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: capture immobilization levels on a antiHis antibody

capture immobilization levels on a antiHis antibody 13 Aug 2014 18:28 #1

Which are the highest capture levels of His-tagged protein that I could reach with an antiHis antibody?
I'd like to study the interaction between a 66kDa protein and a series of small molecules. I need lots of RUs. Is it worthwhile to give a trial? Under standard amine coupling chemistry the protein is not stable.

Thanks a lot
Blanca

capture immobilization levels on a antiHis antibody 13 Aug 2014 20:08 #2

To detect (100 RU Rmax) a 200 Da molecule with your 66kDa protein you need approximately 33000 RU of 66 kDA protein on the chip. To capture 33000 RU of protein with an antibody, you need about 37500 RU of antibody that can capture two proteins. Of course, this is all theoretically.

When you go for less signal (its better), like 20 RU, the amount of antibody to immobilize is less (7500 RU), which is not too difficult to do.

Use a high capacity sensor chip, like the Biacore CM7, Bio-Rad GLH or Xantec CMD500D.
Or try it first with a normal one.

Let us know if it worked out for you!

Arnoud

capture immobilization levels on a antiHis antibody 25 Aug 2014 15:52 #3

Thank you very much for your reply. I've been learning SPR on my own for the last year and your book and this site have been really helpful for me. Thanks a lot for both!
I'm working for a biophysical facility and I need to ask to the users for the materials. If we run the experiment at the end I'll post for sure my results to this site.

Best,
Blanca
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