18 Questions 2 Answers 0 Followers
Questions related from Jamie Henderson
Hello, I have tried to use the no-SCAR CRISPR Cas protocol by Reisch and Prather (2017) on E.coli BL21 (DE3) strain and am struggling to get it to work. I have transformed my strain with the cas9...
18 November 2019 8,903 2 View
Hello, I am currently trying to express the ECD of a transmembrane protein in E.coli. It has multiple disulphide bonds so I co-express it with DsbC and I use a Rosetta-Gami cell line. I was able...
24 July 2018 9,074 2 View
07 July 2018 4,000 3 View
Hello, I am new to working with HEK-293 cells and am having some issues. I was told that as they are loosely adherent in the T75 flasks that I use in order to split them all i had to do was...
18 July 2017 557 5 View
Hello, I'm currently working on two GPCRs in insect sf9 cells and I was expecting quite low levels of expression, which I got. I am unable to find any papers that actually give a value to their...
13 June 2017 3,207 3 View
Hello, I am currently working on GPCRs and am about to create multiple mutants to see their effects on expression level. They are tagged with GFP but am I able to quantify exactly how much...
18 May 2017 6,297 1 View
Hello, I recently performed a BCA and Bradford assay to determine my total protein content following a membrane purification. The two kits gave me approximately 0.89 and 1.1 mg/ml total protein...
05 May 2017 2,061 3 View
Hello, so i am basically doing a time course and seeing the optimal time to solubilise my protein in DDM + CHS. Not really thinking at the time I solubilised my proteins for x hours then when they...
04 April 2017 1,630 3 View
Hello, I am currently working on GPCRs and trying to purify them from the membrane. A big issue I will face in the future is that most other publications use radioligand binding assays as a...
27 March 2017 7,616 0 View
Hello, I am currently expressing a GPCR in sf9 insect cells. I transfected them and performed a protein expression test (western blot) using both an anti-His and anti-GPCR specific antibody. Both...
03 March 2017 5,804 4 View
Hello I am having a bit of an issue with my sf9 cells and trying to express the human PTH receptors. So: I transform DH10 cells which give me blue/white colonies, which I also do a colony PCR to...
28 February 2017 6,362 1 View
Hello, I am currently experimenting on the PTH1R and i wish to tag the N-terminus. I will be using sf9 cells and wanted to see if different signal peptides could increase expression (hemagglutinin...
09 February 2017 1,239 1 View
Hello I need to create a construct that includes the following Maltose binding protein----Protein------6xHis tag I am not sure what the actual process for creating fusion proteins is and would...
06 February 2017 8,537 0 View
Hello, basically I am quite new to PCR and would like some help. I have a vector that I am going to cut with XbaI and HindIII. I want to insert a short signal peptide (15 residues, 45 nucleotides)...
02 February 2017 2,442 2 View
02 February 2017 9,023 0 View
Hello, the issue I am facing is I would like to put a his tag on the N-terminal of my GPCR, but it has a signal peptide for the first 20 or so amino acids and I am unsure of how to get around this...
16 January 2017 6,507 3 View
Hello, as the question says above I would like to know how you go from a cell line to obtaining the structure. I will be using sf9 insect cells. From what I understand so far I will have to...
06 January 2017 8,074 2 View
So I am performing cloning for the first time and i don't think I have done it correctly. The sequence I want to clone is as follows....
01 January 2017 8,440 1 View
