How to avoid protein precipitaion ?

I am working on a thermophilic protein (pI: 7.72) purified using Ni-NTA chromatography (Final buffer: 25mM Pipes (6.6), 300mM NaCl, 1mM PMSF, 10% glycerol, 200mM imidazole). But the protein...

09 October 2016 8,241 24 View

Does cloning vectors (pET series specially) confers toxicity to the gene/protein of interest?

I am working on a thermophilic protein which is not known to be toxic. It was earlier cloned in pET11a and cell death was observed after 2-3 hrs of IPTG (0.1mM) induction. I then cloned the gene...

06 July 2015 751 4 View

Why do I detect two proteins after purification?

While eluting two of my proteins with 250mM of Imidazole, I am getting two different bands (Size of the proteins are mentioned in the Image attached). The genes are cloned in pET22b without Signal...

06 July 2015 1,710 8 View

Is it recommended to incubate Thermophilic genes at 42 degree celsius after IPTG induction?

11 December 2014 3,428 5 View

Why are my non-specific bands coming even after PCR clean up?

I am trying to clone a T.thermophilus gene. I have used 3%/5% DMSO during PCR amplification (using phusion enzyme). Amplification is coming but after PCR purification, and I am also getting...

05 June 2014 2,160 21 View

Why is the ligation of a gene with different vectors not coming?

I have tried cloning of my gene of interest in pET22b and pET28a, for different ratio, but the ligation didn't work (though colonies are coming in some cases). I am pretty much sure that my PCR...

04 May 2014 6,595 14 View

Do you have any suggestions on how to avoid primer dimers after PCR clean up?

When I am doing PCR clean up after PCR amplification (using phusion + DMSO) of a thermophilus gene, and along with the amplified gene, the primer dimer is also appearing. Do you have any...

03 April 2014 1,399 18 View

What is the denaturation temperature used for plasmid vectors (circularised) in PCR? Is it different from 95 degrees C?

I am amplifying a gene from pET11a vector. Is the temperature for denaturation (95 C for chromosomal DNA generally) different for amplifying the gene from plasmid DNA?

02 March 2014 6,215 5 View

Is there any protein expression host designed for thermophilic bacteria? Can it be expressed in E.coli hosts namely Rosetta, C43 DE3 or not?

I am trying to purify a protein from a thermophilic bacteria (gene cloned in DH5 alpha) but I am not getting expression of my protein after IPTG induction. I have tried with different expression...

02 March 2014 2,976 11 View

Which part of the alsA gene in E.coli is bound to the transmembrane domain?

alsA gene has two sub-units and some part of one sub-unit is bound to the TMD (alsC).

10 November 2013 9,633 0 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

Can two EC50 values with different hill slopes be compared?

Hello, I fit a classical dose response curve Y=Bottom + (Top-Bottom)/(1+10^((LogEC-X)*HillSlope)) to my data (not related to inhibitors etc). The model I use is purely qualitative but it fits...

23 February 2021 8,155 5 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

What is the best temperature to coat an ELISA plate?

Hello, I want to control the temperature of my ELISA plate during incubations to get consistent results. In general, what is the best temperature to coat the plate with antibody? I have been...

16 February 2021 2,377 7 View

Is there a way to tell based on appearance which protein crystal should diffract the best?

I have had unheard of success with protein crystallography lately from a super successful protein expression and purification batch. I have attained a lately reproducible vast amount of crystals...

09 February 2021 5,155 5 View

How to measure the concentration of a labeled protein with no tryptophan or tyrosines?

Hey I'm trying to measure the concentration of a protein I've purified and then labeled with Atto 647 flourescent dye. The protein has no tryptophans or tyrosines (although does have...

04 February 2021 4,482 4 View

How to solve error "Cannot find position restraint file restraint.gro (option -r)." on GROMACS?

Hi, I am about to do energy minimization but the above error occurred, I checked my .mdp file and .top file but could not find the problem. TOP file ; Include forcefield parameters #include...

01 February 2021 780 3 View

Why is Fermented Plant Juice (FPJ) always diluted with water? Could it be diluted with other solvents such as rice water?

I am currently making a research paper with my classmates in Philippine Science High School and we are trying to find references that mixed Fermented Plant Juice with other solvents instead of...

17 January 2021 5,099 4 View

Any stable dye to color acrylamide hydrogel beads?

Hi! We are generating hydrogel beads(50-60um) using microfluidics as single-cell beads. To simply all the experiments (especially the centrifuge), I am looking for a dye. The dye should be stable...

14 January 2021 4,383 8 View

How to phosphorylate specific residues on pymol?

Hi everyone, I am using the pyTMs plug in on Pymol to phosphorylate a particular threonine in my protein. I am having difficulty in selecting only one or two residues - it seems I can only...

05 January 2021 230 3 View