How to increase the pH tolerance of an Enzyme ?

There is an enzyme which pH optima is 6.5, we want to make this enzyme working well at pH 4.5, without disturbing its catalytic efficiency for a reason of industrial fermentation process. The...

06 July 2018 8,909 6 View

Is it possible that Km of key residue mutant is similar to wild type enzyme ?

I am getting Km value of a mutant which is similar to its wild type. How it is possible ? The residue is quite important for substrate binding. Is it possible ? please suggest !

10 November 2017 5,476 4 View

Is it fine to mutate some conserved residues in the active site ?

I am planning to make the wild type enzyme pH tolerant (acidic) without affecting the activity of the native enzyme. Here i am thinking to introduce some residues like lysine or ariginine in place...

08 September 2017 1,322 5 View

Which tool is best for showing secondary structure element on the sequence alignment other than ESPRIPT (which can use the pdb file to do it) ?

I am trying to find some tool or server which put secondary structure elements on top of the sequence alignment which ESPRIPT does very well. But is there any other tool which I can use to show...

02 March 2015 712 5 View

Why the protein after Site Directed Mutagensis is not expressed ?

I have done whole plasmid amplication using XT-20 high fidelity enzyme from merck. After induction with 0.3mM IPTG i did not see any expression of the protein.I am suspecting some mutation has...

01 February 2015 3,091 6 View

How can I isolate double stranded DNA from single stranded in a mixture?

09 October 2014 6,666 5 View

Why is the Difference between Rwork and Rfree 10% when refined at 3.2 Angstrom?

I am little bothered that my structure refined at 3.2 Angstrom, have a difference of 10 % without and with solvent contents. The difference is not changing. completeness is 97.6 % at 3.2 Angstrom....

06 July 2014 6,790 8 View

How does one shade the conserved and non-conserved protein sequences after MSA?

I have used a boxshade server, but here I am finding difficulties as it puts sequence numbers here and there after the job. So does anybody have an idea to do the same without any erroneous job of...

06 July 2014 9,086 4 View

What is "Refmac sigma-AA weighted map data"?

06 July 2014 7,131 5 View

How to improve the quality of my crystal?

As I have purified my protein and set crystallization screen and I tried various way to optimize the crystal quality, such as seeding, changing pH, PEG concentration, salt concentration and...

04 May 2014 8,551 10 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