Forum :: Buffer exchange for SPR


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TOPIC: Buffer exchange for SPR

Buffer exchange for SPR 28 Jul 2013 02:00 #1

Hello,

I am going to be working with this protein in the near future, but first I need to do a buffer exchange since it is in DTT, Tris and glycerol. The pI is 5.19. This is only the second protein that I have worked with for SPR, so I am honestly a little unsure (and nervous) about what buffer to choose. Any suggestions?:

www.prospecbio.com/HSPA5_Human_Hi-5_10

Another question... There are 2 Cysteines. Would it be best to immobilize by the amine groups or the cysteines?

Thanks!!!
Claude Bessy

Buffer exchange for SPR 28 Jul 2013 02:00 #2

Hi Claude,

You can dialyse the protein with a small dialyse filter like the MINI slide-A-Lyzer from PIERCE/Thermo scientific.
You can also try to immobilize directly via the amine coupling. By diluting to about 10 ug/ml the concentrations of the TRIS and DTT are probably low enough. Low amounts of TRIS have only a minor effcet on the immobilization.
The glycerol will give a large bulk effect but after each injection you can see how much protein there is immobilized.

Choosing between immobilization technique based on sequence is a difficult one.
You could try the thiol immobilization since there are only two and one is in the begining of your protein. However it will depend on the structure of the protein if this cysteine is free and accessable.

Let us know what your results are!
Arnoud

Buffer exchange for SPR 28 Jul 2013 02:00 #3

As usual, thank you! I will give it a shot.