No Dissociation after pH Scouting / Pre-
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No Dissociation after pH Scouting / Pre-concentration

  • PJM
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7 months 2 weeks ago #1 by PJM
Good morning all,

I hoped to ask your thoughts on some unusual behaviour of a new protein I have been attempting to work with on our 8K.

As you can see in the attached image I did a routine pH scouting run for three similar proteins - 1, 2 and 3. For Protein 1 we saw no interaction with the CM5 chip surface at any of the pH values tested. For Protein 3 we saw great, textbook results.

It is Protein 2 that is most curious to us - we see acceptable, pH-dependent pre-concentration at the surface but then almost no dissociation away after the injection has finished. I've always known this is a situation to avoid but on trying to read up on this in more detail all I have found comes from a Cytiva user handbook - 'At pH 4.0, the sensorgram is irregular and bound material does not dissociate from the surface at the end of the injection, indicating that the protein is aggregating or denaturing.'

I have gone ahead and amine-coupled this Protein 2 to the CM5 chip using pH 4.5 acetate anyway just out of interest - I presumed that perhaps I would still have a useable surface, just with a very low activity level. Compound binding experiments so far have been inconclusive on this however. 

Protein 1 and 2 are the same protein with a pI of 5.40. They are just different batches from the same commercial supplier. Protein 3 is a related family member of Protein 1/2 with a pI of 6.30.

So I suppose my main questions are:
- Are there any further details anyone can share about this lack of dissociation after pre-concentration? How big of a problem is it likely to be for generating a useable surface?
- If the protein is being denatured so quickly by the acetate buffer - do we think alternatives (maleimide, etc.) are worth investigating?
- How likely might it be to get a covalently attached surface for Protein 2? I'd rather avoid something like His-capture with regeneration due to the cost/difficulty of obtaining this protein commercially.

Thanks in advance for any thoughts,
  • Arnoud
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7 months 2 weeks ago #2 by Arnoud
The problem with pre-concentration is the low ionic strength and the lower pH. This can for some proteins a trigger to denature or aggregate. Some proteins renature when back in a ‘normal’ buffer but you need a positive sample to validate the surface (antibody ?).
One alternative is to biotinylate the protein and use a streptavidin sensor surface or the Biotin CAPture kit. This will keep al the proteins in a physiological solution.

Kind regards
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