Dear Earrawandla Janapriya

are you observing dimers in SDS-page?

Do you mean non-reducing SDS page?

If those dimers are present in Non-reducing SDS-page and NOT in reducing SDS-page, you have formation of S-S bonds beetween the different molecules probably due to the progressive oxidation of your Beta mercapto ethanol. Theoretically you can avoid it by replacing B-me with a more stable reducing agent (eg TCEP)

But i suspect that if you have 11 cisteines some of those theoretically can be involved in S-S bond and with the addittion of the reducing agent you are destroing it and induce protein unfolding.

Have you never tried to perform lysis and purification with-out B-me? Was the protein non soluble with out B-me?

best regards

Manuele

TCEP is a much more stable reducing agent, maybe try that? Good luck!

You may treat the cell lysate with Dithiothreitol (DTT). DTT helps to reduce protein disulphide bonds, which can reduce the dimers. The concentration of the DTT depends on the predicted disulphide bond in the protein. The amino acid sequence of your protein should give you information on the number of these disulphide bonds. You can also predict the disulphide bond in your protein using; http://liulab.csrc.ac.cn:10003/predict.

As many recommended, reducing agents like DTT or TCEP can certainly help you. There is only one problem with that. Ni-NTA does not really tolerate reducing agents. 1mM is the maximum I think.

May I suggest INDIGO-Ni beads instead? (using the same protocols).

It has 20 times higher DTT tolerance by roughly the same protein yield as Ni-NTA beads. This way you should be able to use reducing agents without worry.

https://cube-biotech.com/indigo-features

The page I linked also has a form to request a free sample.