Is it possible that you cannot detect your peptide on the chip because it is to small?
Lowering your buffer below pH of 3.0 will not help since the dextran matrix will loose its preconcentration ability. But injecting your peptides at pH 3.1 - 3.3 (10 mM buffer, no salt) should still work.
I tried using various pHs - from 4.0 through 3.0, 2.0 and 1.5. Also tried various buffer and peptide concentrations. I could not get any binding.
I do not believe the size is a problem. Peptides should be readily detected if they bind.
It is as if the negative charge of the peptides does not enable them to get close to the streptavidin on the chip and bind through their biotin...
I thought of a second round of EDC/NHS activation and then ethanolamine in order to reduce the overall negative charge of the chip (less COO-). But I do not know how this will affect the bound streptavidin.
I'am not in the structure of Streptavidin, but I really don't think the charges on the chip are the problem.
I you want to do it, do it before coupling the streptavidin or use a other chip with less COO- groups.
Easy solution is to buy a streptavidin chip and check the biotinylation.
Hi Arnoud, I really appreciate this discussion... It really helps me.
I've already used these peptides previously on streptavidin beads and they bound with no problem. We also check peptides by Mass Spec after synthesis and HPLC cleaning and verify that the biotin is there. I don't believe the biotin is the problem.
As far as I know the ready-made streptavidin is the CM5 chip with strep bound to it (in the same way we do it but much more expensive). So that wouldn't help much
I am sure that the streptavidin is bound because it its binding was detected and stable...
I thought of checking that the strep is OK by trying to bind something that has previously worked. This will waste a lane but at least help understand what's wrong.
Trying to do another round of activation wouldn't matter much since we would probably not use this chip for other things anyhow...
Another solution is to get a new chip and bind my protein (50 kD) then use the peptides (1.5 kD) as analytes. But I understand that the signal would be too weak for good analysis. Is this true?