Can anyone advise on now to compare ligand binding sites in NMR and crystal structures?

New to crystallography...I want to compare ligand binding in a crystal structure to one generated using NMR. I have tried the pdbsum and the pisa servers but the hydrogen bonds that are identified...

11 December 2016 3,924 3 View

Does any one have any experience of creating a stable cell line using Calu-3 cells?

I am looking to create a stable cell line using Calu-3 cells. My initial plan is to co-transfect with pBABE-puro and select with puromycin. Does anyone have any similar experience with Calu-3? I...

07 August 2016 2,513 5 View

Do I need to codon optimise my sequence for expression in a mammalian cell line?

My POI is a human protein. However we when we had the DNA sequence synthesized we codon optimised for S.cerevisae (for protein expression). However, I now wish to do some interactome studies in a...

06 July 2016 5,892 4 View

What's the difference between the AKTA prime and AKTA prime plus system?

02 March 2016 5,380 5 View

How do I determine the protein concentration of a peptide?

I am working with a construct containing only the C-terminal region of my POI. It is 42 residues long, and contains no aromatic residues (but does contain a number of basic amino acids). Following...

10 November 2015 9,840 4 View

Does anyone have any advice for tag cleavage with enterokinase?

03 April 2015 7,991 3 View

Looking for Materials with high thermal expansion coefficient and high temperature resistance?

03 March 2021 8,272 1 View

How to grow E.coli on the low salt LB agar plate with 50 or 100ug/ml of Blasticidin?

Hello, I would like to ask how to grow E.coli which resists 50 or 100ug/ml of Blasticidin. I have tried many methods by using low salt Luria-Bertain(LB) agar plate. However, no colonies grow. Let...

01 March 2021 2,027 2 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

Are there lab markers/pens resistant to Roti-Histol?

Hi, I'm looking for a pen/marker to write on the smooth, transparent (non-frosted) part of microscopy slides which does not get washed away by alcohols and most importantly Roti-Histol. I want to...

24 February 2021 9,902 5 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