How long do I need to treat transfected HEK 293 cells with Brefeldin A and the concentration need to see endo H resistance in glycosylated protein ?

More Neeraj Sharma's questions See All

Is my signal sequence is correct or functional?

My cloned gene contain a signal sequence at N terminal . How can I check the nucleotide sequence is a correct signal sequence. The following uppercase letter are signal sequence cloned in...

01 February 2019 7,597 2 View

Could any one please provide me a mammalian expression vector with AviTag to add at C and N terminal of protein ?

I want to add AviTag with my protein at N and C terminal and to express it in mammalian cell line.

09 October 2018 6,660 0 View

Can I get single fragment using two PCR product in single reaction ?

I am planing to get two gene together for cloning purpose. My plan is first amplify the gene "x" using a set of primer which will amplify gene x without stop codon. I will amplify another gene "y"...

08 September 2018 6,654 1 View

Can I preserve my Transfected HEK-293 Cells IN -80 degree for few days, Later these will be being used for Microsome Isolation?

08 September 2018 8,512 1 View

What will be size of my expressed protein If my construct contain Mut. Htt96Q linked with BiRA* ?

My construct contains Htt 96Q followed by BirA* with a 6 amino acid linker between them. Than what will be size of my expressed protein if molecular mass of BirA* is 35.2 KDa? Based on amino...

07 August 2018 3,656 1 View

Why I found colony for negative control (contain only double digested vector : one blunt and one sticky end)after transformation in DH5-alfa cells ?

Why I found colony for negative control on selection plate (contain only double digested vector : one blunt (by EcoRV) and one sticky (by SalI) end with ligation mixture, no insert, after...

06 July 2018 231 1 View

I want to ligate my 1kb insert (PCR product) with 6 kb vector, both are digested by ECORV (creating blunt end) and Sal1 (creating sticky end). How?

I want to ligate my 1kb insert (PCR product) with 6 kb vector, both are digested by ECORV (creating blunt end) and Sal1 (creating sticky end). How? Is blunt end may create problem in cloning?

05 June 2018 6,728 3 View

Why Polyst. Flat bot. high Bind. MP (Costar) blank micro-plate is reactive with positive control?

I am using Polyst. Flat bot. high Bind. MP blank (Costar) micro-plate for making ELISA (HIV ), when i am using this blank plate for ELISA assay , it is found reactive with positive control while...

31 December 2016 5,725 6 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

What is a good reagent for lentiviral plasmid transfection in HeLa cells?

I have been using Lipofectamine 2000 but was hoping there may be a better protocol or one for something like Lipofectamine PLUS reagent.

24 February 2021 1,633 4 View

Why the cells die after stable transfection?

I tried stable transfection in the U87 cell line with the sgRNA-Cas9 All-in-One lentivector. Cells were cultured in a 6-well plate, 4ug plasmid was transfected after 24hs when the cell confluence...

22 February 2021 2,864 4 View

Is there a method for evaluating nanoparticle adhesion to epithelial tissue in vitro?

I want to measure how well nanoparticles can transfect cells based on NP adhesion properties. In the literature I find people do it in animal studies, and epithelial adhesion helps in transfection...

21 February 2021 7,791 3 View

Are exogenous molecules transferred between human cells during doubling ?

Hello RG community, I wonderer whether exogenous molecules (e.g. reporter plasmids, miRNAs or other artificial molecules) are transferred between cultured human cells during their doubling ? So,...

21 February 2021 6,633 3 View

Which transfection reagent is the best for plasmid transfection of MDA-MB-231 cell line, Lipofectamine based or other reagent suggestion?

I try to transfect MDA MB 231 cell line with a plasmid by PEI, unfortunately, the rate of transfection is low, and now I wanna find and order the best transfection reagent for this cell line and...

19 February 2021 828 6 View

Jürgen Denecke

5 micrograms per millilitre Brefeldin A in your cell culture and incubation for 1 hour will be enough to get mixing of Golgi and ER membranes, so your protein will acquire endoH resistance if it is competent for complex glycan formation. I recommend to do it when your protein is well expressed (i.e. 24-48 hours after transfection). If you add Brefeldin A immediately after transfection, expression levels will be reduced.

Neeraj Sharma

Thanks Denecke.