I am using a magnetic bead protocol which involves a step of non-denaturing elution to remove bead-antibody from antigen. The protocol calls for 20ul 50 mM glycine pH 2.8, and I have been using 400-800ul plasma. I want to normalise the pH for subsequent immunoassay and the protocol suggests "...the pH of the eluate can be adjusted by adding 1 M Tris, pH 7.5".
Does anyone have experience of doing this with accuracy in such small volumes?
Thank you for your help.
When I'm trying to adjust the pH of small volumes and need to monitor it, I use test strips and pipet a microliter of my sample onto the strip (I use test strips with three segments, and I pipet the droplet onto the boundary between the two segments that are going to be giving the colour change that is most informative with respect to the pH I'm shooting for).
So you take a couple of microliters, put it on the test strip, adjust the pH, and do it again. It usually takes a few tries to get the pH you want, so you wind up using 3 or 4 microliters of your sample.
Since your Tris is pH 7.4, you can't overshoot. Just add a 5x molar excess and take it on faith. If you want, you can optimize this on large scale with a pH meter, and then multiply all the volumes down by a constant factor. (but you don't really need to)
What John Schloendorn suggested is the easy way. When purifiying IgG from serum on Protein A gel, I elute with 0.1M glycine/HCl, 0.5M NaCl buffer, pH 2.8 and for each 1mL of eluant I neutralise with 0.2mL of 2M TRIS/HCl, buffer, pH 7.5
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