Hi Robert, I started a thread on RG about inclusion bodies. There are some comments there that might help you.

https://www.researchgate.net/post/Why_are_so_many_researchers_trying_to_isolate_proteins_from_the_tangled_mess_of_coli_inclusion_bodies

Here is a review of the subject, that includes methods for cleaning up inclusion bodies.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379949/

Hi Robert,

What do you mean by 'high level of impurities'?

If you mean something carried on from the cells into the inclusion bodies, I recommend you use Cell-lytic B to lyse the first pellet (after you have lysed the cells and spun them down), to lyse any cells which were not lysed before.

Second, the usage of DNase is very critical, because if you do not get rid of the DNA, it will bind to a lot of proteins, thus the impurities.

Third, it is vital to be generous with DTT in all stages of the purification/washing of inclusion bodies.

Finally, when you run your SDS-PAGE gel, make sure you have boiled your sample and added enough reducing agent prior to gel loading. Especially, if your protein form many disulphide bonds.

Hope this helps.

Good luck.

Thanks Stefansson, thanks Shapiro, thanks Halabi. Let me see what I can get.