Many Thanks Artem. I will use Kifunensine in culture for next batch expression, and will use EndoH with already purified proteins.
Best Wishes, Partha On Mon, Jul 16, 2018 at 12:26 PM, Artem Evdokimov <artem.evdoki...@gmail.com > wrote: > Dear Partha > > Treat your culture with Kifunensine prior to transfection (or throughout > growth if you're using stables) and then treat purified protein with EndoH. > Pretty cheap and effective. > > PNGase F does not *require* denaturing conditions. It just likes the > protein to be 'loose' - in my experience about half the time the accessible > sites will fall off even under native conditons. However, PNGase F is > somewhat expensive and after treatment certain proteins have fallen out of > solution (presumably because not a single carbohydrate remained, as opposed > to 'shielding' provided by the single remaining sugar left behind by > EndoH). Caveat emptor. > > Artem > > - Cosmic Cats approve of this message > > On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar < > spart...@gmail.com> wrote: > >> Dear All, >> >> I am in a situation, almost for the first time within my limited >> experience, that deglycosylation might be necessary to obtain crystal. So, >> I thought of tapping to vast experience of CCP4BBers, while I am searching >> literature. >> >> I have protein that has been expressed in HEK293 cells, secreted into >> media, purified over IMAC and SEC columns. Crystallization-screens with its >> binding partners (they form good complexes based on analytical SEC) have >> not produced any useful hits (whereas complexes with related proteins >> worked well). So, I plan to re-try complex formation and \crystallization >> screen after deglysosylation. >> >> My question is: In practice, Does a kit (for example here: >> https://www.sigmaaldrich.com/catalog/product/SIGMA/ >> NDEGLY?lang=en®ion=US) containing Endo F1, F2, F3 be sufficient or >> should this be tried in combination with PNGase (which requires >> desaturating conditions)?!! >> >> Many Thanks in advance for your suggestions, and reference. >> >> Best Wishes, >> Partha >> >> ------------------------------ >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >> > > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
