Adding natural ligands often improves protein stability.

Hi Shatanik, it depends on the protein. You can try adding substrate or inhibitor, also try with phospholipids like DPPC/DOPC , Asolectin etc.

Best

Edwin

the point is the protein shows a progressive degeneration over days after purification. I have a DDM/CHS misture for solubilization. Adding natural ligand is not an option.

Dear Shatanik:

Have your tested different DDM concentrations? Please, have a look to this paper. It may help

https://www.nature.com/articles/srep41751

Good luck,

Rosa María Martínez-Espinosa

Probably your protein is missing some component, You can try different DDM concentration as Rosa Maria mentioned also adition of cardiolipin during purification can be a good option.

Article Role of cardiolipin in stability of integral membrane proteins

Best

Edwin

How are you storing the protein after purification? I would recommend storing it flash-frozen in single-use aliquots at -80 or in liquid nitrogen.

Some proteins have limited stability in the solubilised state. Days is actually good, it could be minutes :-(. One reason can be the loss of essential (anular) lipids. For this reason, you need to maintain the [detergent] at precisely the cmc, which means that you need to determine that for your buffer and temperature.

You could also try to add osmolytes like glycerol, sugars and their alcohols, TMAO, sarcosine, PEG and others. These reduce available water concentration and hence protein flexibility. This reduces enzymatic activity, but also denaturation. Also, osmolytes increase surface tension. Since denatured proteins have more surface area, the native state is favoured in their presence.

I assume that you work under sterile conditions, so that bacteria cannot degrade your protein?

Thanks Engelbert, Yep I work under sterile conditions so it would not be bacteria, but I could increase the stability from days to weeks by using an osmolyte mixture. Although would be really helpful especially for crystalization to completely not have this annoying problem.