Asrar Alam try such one: 50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM Na4P2O7 supplemented with 10 μg/ml Aprotinin, 1 mM PMSF, 2 mM Na3VO4, 10 mM NaF.

Thank you Yuri Cheburkin

Dear Asrar Alam

whay you did not add an n-terminal signal peptide to your protein sequence to secrete it into the medium? In this way you can direclty recover it by IMAC with higher purity since the amount of extracellular protien is certanilly low.

best regards

Manuele

In case you are interested in the following link

https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html

you can find some guidelines to perform IMAC from the surnatant of Expi293 that are similar to HEK293 growth in suspension.

best regards

Manuele

Thank you Manuele Martinelli . Indeed it will be much easy to purify the protein from the supernatant. Unfortunately, my constructs do not have the leader sequence for secretion. Perhaps, they can be used for other purposes also like pulling out interacting partners.

Dear Asrar Alam

add a signal peptide on your construct is probably much eaasier than oprimize the purification fron total extract

in the following link you can see an example

https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html

best

manuele

Thank you Manuele. I will go through your blog.