Forum :: Help - biotinylated peptide does not bind to CM


Welcome, Guest
Username: Password: Remember me
These are the posts of the old forum. It was not possible to transfer the user data, so they are missing in most of the posts. For new questions, go to the general discussions.
  • Page:
  • 1
  • 2

TOPIC: Help - biotinylated peptide does not bind to CM

Help - biotinylated peptide does not bind to CM 14 Nov 2006 01:00 #1

Hello,

I am trying to measure an interaction between my protein of interest and two peptides.

These peptides were biotinylated but did not bind my Streptavidin-coated chip (A CM5 chip activated and bound with streptavidin, then deactivated with ethanol-amine).

I believe the problem lies in the low pI of my peptides.
One peptide has a pI of 3.6 and the other 4.0.

I tried injecting with glycine (pH=1.5) as buffer but did not detect any binding.

Any ideas or solutions?

Thanx.

biotinylation 14 Nov 2006 01:00 #2

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.

Tried that already 14 Nov 2006 01:00 #3

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.

Any thoughts?

biotinylation 14 Nov 2006 01:00 #4

It is obvious but dit you check your biotinylation. It can be done with a procedure form Pierce

www.piercenet.com search for HABA.

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.

I hope I'am of help

Arnoud

biotin is good 14 Nov 2006 01:00 #5

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?

detecting low mass 14 Nov 2006 01:00 #6

Because SPR is "mass based" you can calculate what you can expect.

Immobilization of a 50 KDa ligand and detecting with a 1.5 KDa analyte. How much to immobilize for a acceptable signal?

So calculate (Mwligand x Rmax) / (Mw analyte * valency)

e.g: (50.000 Da x 100 RU ) / (1500 Da * 1) : immobilize at least 3500 RU ligand.

This is theoretical because some ligand molecules will be inaccessible etc. etc.

It is worth a try.

Arnoud
  • Page:
  • 1
  • 2