Dear Ralf Kölling

There is an affinity of Phos-tag toward the abundant carboxyl groups of acidic proteins. For instance, the negative charge of the carboxylate group of glutamate is thought to mimic the negative charge of phosphate resulting in Phos-tag binding.

You may want to refer to the articles attached below.

Article Specific glutamic acid residues in targeted proteins induce ...

Article Phos-tag-based phosphate affinity chromatographic techniques

Some useful tips.

It is generally recommended to omit EDTA from lysis and sample buffers as it chelates Zn2+ ions from Phos-tag gels. Some phosphatases or protease inhibitor cocktails do contain EDTA. If it is not possible to avoid using EDTA, add equimolar amounts of ZnCl2 to the sample buffer. Avoid using phosphate-containing buffers, such as PBS during lysate preparation (e.g. washing steps) as phosphate ions can bind to the Phos-tag complex which would result in less efficient separation of phospho-proteins.

Regards,

Malcolm Nobre

Malcolm Nobre

Thanks for your comment. This is exactly what I was looking for, but couldn't find. There is a EADD sequence in my protein, which could be a potential candidate for Phos-tag interaction. I will test this by deleting these residues. But, it is strange, why such a sequence, which occurs in almost all proteins, leads to an interaction only in some proteins. There must be something peculiar about the SDS association in these proteins.