Forum :: Other buffer problems


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TOPIC: Other buffer problems

Other buffer problems 05 Mar 2007 01:00 #1

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

Other buffer problems 05 Mar 2007 01:00 #2

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
Last Edit: 13 Mar 2015 20:30 by MQAdmin03.

Other buffer problems 05 Mar 2007 01:00 #3

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

Other buffer problems 05 Mar 2007 01:00 #4

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