Rmax is variable ?

hello everyone,

My question is how can i control Rmax... I get Rmax anywhere between 95-675 RU... this is across several runs. i have immobilised my receptor FcgRIIIa at (aim for 2000RU) and run my antibody in several dilutions..

just wanted to ask is there a way in which i could control the Rmax.. I understand that it is the analyse binding capacity but why does my analyse binding capacity vary so much across different chips and runs? what am I missing?

any help would be great!
Thanks
Best regards
Biaenthu

BTW I use BiacoreT100 and CM5 Chip in HBSEP buffer (running as well as dilution buffer)

It is not totally clear to me what you mean. Therefore some questions.
1) when you immobilize FcgRIIIa, how much do you actually immobilize?
2) the rmax you state, are your curves go that high or is it the Rmax in the fitting?
3) how big (kDa) are the ligand and analyte?

In addition you could read www.sprpages.nl/immobilization/immobilization-procedures.html for extra information.
Arnoud

1) We immobilise 2000RU
2) yes curves do go high, and close to the obtained Rmax
3) ligand is FcgRIIIa (50kDa) and analyte is 150KDa

Hope this helps to clarify the question. and i am sorry for the late response

A quick calculation for the Rmax gives (2000 x 150)/50 = 6000 RU for the binding of the analyte to the FcgRIIIa. This is theoretical because both ligand and analyte must in these cases 100% active, what is in general not the case.

If you inject the antibody you say that the curves go almost to the fitted Rmax. When you look at the kinetics and calculate the KD, is the highest injected analyte concentration more than 10 times the KD? Only at 10x KD the curves will approach the Rmax.
You can read for instance www.sprpages.nl/sensorgram-tutorial/a-curve.html to learn about the relation of analyte concentration and Req and Rmax.

You say that you have large differences in Rmax. . I can imaging that you use a regeneration which can be damaging the ligand or not strong enough to remove the analyte. Did you monitor the baseline before the analyte injection and after the regenearation?
If possible can you post one or two sensorgrams?

Arnoud

yes i would post a sensogram...also, for this method we have not used any regeneration...