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Questions related from Harry birdy Austin
Hi everyone, I'm currently conducting protein expression trials with a difficult to express enzyme. I'm trying a range of induction times, temperatures and induction OD. I notice that at a lower...
03 March 2019 387 5 View
Hi everyone, I know I'm asking a lot but I'm learning HPLC for the first time. I'm working with a simple assay containing three simple compounds that separate well. I've recently inject standards...
03 March 2019 3,241 4 View
Hey everyone, I'm working with an esterase at the moment and I want to preform a pnp-acfetate assay. I'm just curious as to how people here prepare the stock solution of PNP-acetate. Basically...
11 November 2018 6,397 3 View
Hi everyone, I'm sequencing a gene post site directed mutagenesis and so far I'm getting horrible results with poor coverage. For the same gene I sequence twice using the T7F and T7R primers. I...
06 June 2018 8,957 3 View
Hi everyone, I'm trying some site directed mutagenesis at the moment and having problems with the sequencing. I'm new to sequencing so I thought I'd try to get some tips. I'm currently using...
01 January 2018 753 2 View
Hi guys I'm looking to use SDM to change a single residue in an enzyme I'm working with. I'm new to this so I thought I would ask advice. Is there a current protocol that works best or should i...
11 November 2017 692 5 View
Hey guys I'm working with PNP-acetate and PNP-butyrate, two model substrates for esterase/lipase reactions which yield a product which absorbs at 405 nm on hydrolysis. All of the assays I've...
10 October 2017 9,148 5 View
Hey guys I've been using the Qiagen gel extraction kit to purify my DNA post restriction digest. I seem get very low yield but my big problem is the purity of the DNA product. When I spec it I get...
06 June 2017 1,511 4 View
Hey guys, I've recently cloned a protein into a vector and attempted expression. Colony PCR and gels confirmed an insert of an appropriate size was in the colony that I selected for protein...
06 June 2017 217 5 View
I think there should be. We could have one for each category. So researchers put in a considerable amount of time to helping people with their research. What does everyone else...
03 March 2017 580 0 View
Hey guys I'm trying to knock out a gene in a fast growing species of mycobacterium called M.smegmatis. The techniques I came across involve one and two step recombination techniques with pGOAL and...
03 March 2017 1,253 25 View
Hi guys, I want to knock out a specific enzyme in a species of mycobacterium. I have no prior experience with this and I was wondering if anyone could point me in the right direct? Is there a...
03 March 2017 6,049 2 View
Hey guys, I'm working on protein that usually expresses well. Previously I've been getting it to express completely soluble and in milligram quantities, however lately I've been having problems...
02 February 2017 3,499 3 View
Hi guys, Some of my recent protein crystals have turned into sphericules. These were small crystals to begin with but they have appeared to turn into oil form. Is this common, and is there...
01 January 2017 8,320 4 View
Hey guys I'm trying to isolate a tightly but not covalently bound co-factor from an enzyme I'm working with. I'd like to do it without reducing the co-factor if possible. Is there any mild...
12 December 2016 8,914 4 View
I'm working with a tri-substrate system and want to gain insight into the the enzyme's mechanism and the relationship on substrate concentration and rate. I plan to use a microplate assay where I...
11 November 2016 8,439 3 View
Hey guys I'm working with an extremely flexible multi-domain protein which has proved highly resistant to crystallization. I've tried a variety of concentrations, protein-ligand concentrations and...
11 November 2016 4,888 4 View
Hi I'm working with an enzyme which appears to have some degree of product inhibition. It has a very fast initial rate and a huge dip in rate followed by a steady rate. I suspect this may be...
11 November 2016 9,708 4 View
I'm working with a protein that forms different complexes. This is evident during a size exclusion run but lately, during the elution stage of a metal affinity chromatography run, the protein...
10 October 2016 4,226 3 View
Hey everyone, I'm working on an enzyme which utilities three substrates. One is NADH and the others are ATP and another substrate. I'm following the oxidation of NADH...
09 September 2016 5,797 10 View
The absorbance at 340nm indicates that my protein binds to NADH or NADPH but how would I confirm that? I haven't got access to mass spec at the moment but was thinking of using a nucleotide HPLC...
08 August 2016 8,791 4 View
My protein is quite a large one and in theory should be easily separated by SEC. It does and I get quite pure fractions. The thing is my protein elutes in several different peaks i.e sizes. I...
05 May 2016 5,304 7 View
Hey everyone, I'm working with a protein that I managed to purify to ~95% purity as estimated by SDS PAGE. The protein seems to be stable for at least a week however after that time it loses...
04 April 2016 2,417 3 View
Hey guys I couldn't find much literature on this. I'm working with an enzyme that may be covalently attached to coenzyme A and I'm looking to determine if A: the cofactor is present and B: if so...
03 March 2016 6,506 5 View
Hey guys I will soon be conducting enzyme assays with a series of fatty acids of varying solubility. Has anyone got any advice on how to solubilise these for kinetic studies without increasing the...
01 January 2016 5,348 3 View
Hey guys I'm attempting to express quite a large protein in XL1-Blue cells. The protein's approximately 140KD . It is a bacterial protein but not one found in E.Coli. I have seen evidence (SDS)...
11 November 2015 2,528 7 View
Hey guys I finally have some very small crystals of a flexible multi-domain protein I am working on. The problem is that they are far too small to produce diffraction data, with the smallest...
01 January 1970 8,154 1 View
