Other buffer problems

Hi everybody reading that,

I'm just about to do my first SPR experiment (on Monday), but the method was only briefly introduced to me. And so, one important question arose: what kind of buffer may I use as the flow buffer?

Few things: I wanted to use sodium acetate for ligand that's going to be immobilised. Then, my sample is in the Tris buffer. And my activating solution is in MES. Now, can I use the acetate as my flow buffer? Or shall I rather use Tris? And - in any case - won't these buffers interfere with the immobilisation?

Thanks in advance for any help.

Regards,
Rafal

Dear Rafel

I would suggest to read first more background on the subject before attempting to experiment.

Don't work harder but work smarter!

Go to www.sprpages.nl/downloads.htm

download all the files and read today!

Kind regards
Arnoud

Hi once more,

Reading - that's what I'm doing since more than a week. And believe me - I've already read through all the stuff from that page. It might only be that I haven't understood something.

Anyway, I just thought that it would be better if the flow buffer was the same as one of the others buffers I'd like to use - or have to use, because I can't help e.g. having my sample in Tris. But it seems that none of them is really apropriate for NHS/EDC coupling, so I suppose I'll have to stick to PBS. Hope it will work.

It will be what it from the beginning had to be - try and error research.

Thanks anyway.
Regards,
Rafal

Dear Rafal,

In general it is important to have your ligand (the one you couple) in a buffer without reactive groups like amines. So use PBS, HEPES but not TRIS or a buffer with azide.

Coupling under low salt and low pH 3.1-5.0 is the best. Say 10 mM Acetate buffer and 25 - 100 ug/ml ligand.

Second, after immobilizing you can switch buffer to one more suitable for your experiments. We couple with PBS/Acetate and switch to Tyrode for the experiments.

good luck with your first steps.

Kind regards
Arnoud Marquart