Hi Radu,
Many Thanks for your clarifications, and reference for background
materials. I will implement all suggestions in my next expression batch.
Best Wishes,
Partha
On Mon, Jul 16, 2018 at 1:53 PM, wrote:
> Hi Partha,
>
> In this case (un-treated HEK293), you cannot use EndoH with already
>
Oops,
I made a mistake in my previous email, apologies! Griffithsin would also
require high-Man glycans, so kifunensine treatment or GnTI-/- cells So
yes, it's either PNGase or cryo-EM...
Best wishes,
Radu
> Hi Savvas,
>
> Many Thanks for your inputs and references. This cell line used is
Hi Partha,
In this case (un-treated HEK293), you cannot use EndoH with already purified
proteins (as per your reply to Artem's email). The N-glycans will be
fucosylated, hence EndoH-insensitive (see PMID: 23623336 for background
infos). You can still use PNGase. But, as Artem suggested, this often
Hi Savvas,
Many Thanks for your inputs and references. This cell line used is not
HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
cell-line stably expressing (created using lenti-methods by a
former colleague) the protein of interest. In future, I will plan to do
expression in t
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 wrote:
> Dear Partha
>
> Treat your culture with Kifunensine prior to transfection (or through
Dear Partha
you do not specify which HEK293 cell line you have used, but if it so happens
that it is the very handy HEK293S MGAT1-/- cell line (previously known as
HEK293S GnTI-/- ) which produces N-linked Man5GlcNAc2 glycans you might want to
consider using EndoH (e.g. see Verstraete et al. DOI
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 experienc
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, secr
