For the protein to be stable and functional, which is the best pH of the buffer?
Hi all I ligated my small insert (37bp) into 3.5kb plasmid, I got colonies but when I check the sequence of my insert, I found more than one insert ligated as a repeats, some clone got three...
01 February 2019 4,120 6 View
I transformed 4.5kb plasmid with Ampicillin marker into DH5α by electroporation, I need to get the actual number of transformed bacteria on the plate (i.e. one plasmid per bacteria), after...
07 August 2018 5,941 5 View
Anyone know a suicide plasmid that can be used to delete a gene in E.coli strain? I need to use a suicide plasmid to delete a gene in E.coli strain. I used one for gene deletion in Pseudomonas but...
11 December 2017 257 1 View
Dear Dave I am working on a protein (around 125KD) and I have got some mysterious things regarding the purification so I need your help. The protein was expressed using pET28a vector in E.coli...
06 July 2015 6,930 18 View
I have purified a protein which suppose to adenylate amino acid consuming ATP and releasing AMP and PPi, so to test the protein activity I have to measure the released PPi in the solution. Can...
08 September 2014 8,888 18 View
I am going to design two oligonucleotides (the size about 20 base) which can be annealed together forming two sticky ends then will be ligated to the digested vector with the corresponding enzyme....
03 April 2014 8,015 1 View
I am going to extract protein from BL21 but I do not know how much of EDTA-free protease inhibitor (tablet from Roche diagnostic) should I use.
31 December 2013 10,073 1 View
Does anyone know how to add Flag-tag to the C-terminus of the protein? What is the principle of the procedure? I am planning to transform bacterial gene to mammalian cells and I have to check the...
31 December 2013 2,517 6 View
I'm working on selecting antibodies against a recombinant protein that has a His-tag. My idea is to first bind the recombinant protein to a HisTRAP column and then use this column for an affinity...
07 August 2024 505 3 View
Is it the "elution buffer" or the "dialysis buffer"? Note: I'll be using NanoDrop OneC
01 August 2024 967 3 View
I'm having problems with ion exchange chromatography. After applying my sample (a fluorescent protein), a preferential path inevitably forms. The matrix I use is DEAE-Sephacel. Could someone...
27 July 2024 6,605 0 View
I use 0.5% sarkosyl to solubilise recombinant proteins contained in Inclusion bodies of E. coli. sarkosyl very effeciently solubilizes proteins from Inclusion bodies and I am trying to use...
25 July 2024 5,001 2 View
"The Correlation between Nutrition and Transport Mechanism under Abiotic Stress in Plants: A Comprehensive Review" by Muhammad Saleem, Jianhua Zhang, Muhammad Qasim, Rashid Iqbal, and Li Song,...
23 July 2024 9,109 0 View
Good morning, I am trying to produce a recombinant protein. Unfortunately I one of the two restriction enzymes that I use do not cut at the end of the MCS. After the digestion 15 nucleotides of...
17 July 2024 2,787 4 View
My protein is a transmembrane protein, therefore i need to construct a transmembrane using CHARMM-GUI. Can someone please suggest me a tutorial for the same.
24 June 2024 5,044 1 View
Hi, may I know I want to fuse my protein with affinity tag for protein purification in Expi293, how can I determine and visualise how does a N or C terminal tag affect my proteins (structure,...
24 June 2024 8,622 6 View
Hello esteemed scientists, I am endeavoring to increase the yield of my recombinant protein. It is a 120 kDa protein tagged with MBP and features three intrinsically disordered regions with pI...
12 June 2024 9,616 8 View
Hi everyone, I've been having difficulty in purifying a protein. It is a beta barrel, integral membrane protein. My protein is expressed and purified recombinantly. My target protein is running...
30 May 2024 7,604 3 View