Acidic Proteins - Amine Coupling?

Hello all,

Currently working with a protein (kinase) that has a pI of 5.0 and wanted to double check all of the options open to me.

It amine couples perfectly well to CM5 chips using low pH (4.5 / 4.0) acetate but I see no binding response from a number of well validated tool compounds - I'm assuming because it is a kinase domain and a literature search suggests this low pH will have caused the loss in activity.

I've seen an example to get around this by successful amine coupling in less harsh conditions (sodium borate, pH 7.8 in this case) but for my kinase I would be well above the pI - meaning a net negative charge on the protein. Is amine coupling at all feasible under these conditions - with the 'repulsion' away from the chip surface preventing any pre-concentration?

Happy to hear any thoughts on this and thanks in advance - my Plan B and C is to try biotinylation of the protein or else an affinity-tag capture approach but keen to see whether any amine coupling ideas can be tried first.

 

Amine coupling also works with a pH equal or higher to the pI but is less efficient. You need higher ligand concentrations and longer contact times to get the same immobilization level. Options B and C have the benefit of using physiological pH conditions.

I would like add option D. By incubating the ligand with an excess of analyte the active site of the ligand will be protected during immobilization at a lower pH. This only works with small analytes that will not immobilized during the amine coupling
.
Bear in mind that the amine coupling in general uses a lower pH than physiological conditions but also lacks any salts.

Kind regards
Arnoud

Hi Arnoud,

Thanks for the helpful response. Funnily enough I did try option D.) just after writing this post - I amine-coupled the protein in the presence of 1 uM inhibitor (low nanomolar literature Kd) and it seemed to immobilise slightly better. The binding response of the tool compounds looked slightly improved but still nowhere near where I would expect, based on similar literature nanomolar affinities.

So - I tried the physiological amine coupling (rather than acetate, the protein was diluted in running buffer - HBS-P+) and this was a complete failure - see attached image (brown sensorgram - less than 100 RU immobilised).

I then turned to a capture approach - the second image shows amine coupling of an anti-Flag tag antibody (purple sensorgrams, both flow cells) and I think I near enough saturated the CM5 surface (note reduced RU immobilised in the second image) succesfully with antibody. The third image shows my best result from capturing the Flag-tagged protein - only 5 RU is apparently captured!

So all in all, nothing looks like it is close to working right now - so any thoughts or advice would be greatfly appreciated!

So, this comes down to the validation of the flag-tagged protein or anti flag-antibody. To validate the anti flag antibody, you can inject something commercial with a flag-tag or a know sample that has worked (in ELISA or blot). Second you must prove that your protein has a flag-taq. Coating your protein and detecting the flag-tag in an ELISA set-up is the easiest way. And very basic, did you have your protein on gel? Is it pure and running at the right molecular weight.
As an alternative switch to biotinylation etc…

Arnoud