48 Questions 34 Answers 0 Followers
Questions related from Prem Prakash
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...
07 July 2018 8,961 6 View
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 !
11 November 2017 5,533 4 View
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...
09 September 2017 1,373 5 View
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...
03 March 2015 767 5 View
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...
02 February 2015 3,139 6 View
Please suggest any protocol which is cost effective and feasible. Thanks in Advance.
10 October 2014 6,825 5 View
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....
07 July 2014 6,879 8 View
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...
07 July 2014 9,145 4 View
I am creating a file for the ligand by generating the omit map in order to visualize it into pymol. Does anyone have any explanation what the Refmac sigma-AA weighted map data is, and why it is...
07 July 2014 7,181 5 View
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...
05 May 2014 8,607 10 View
What does it actually imply? I am attaching the chart came after refmac 5. Can anyone please give an explanation of its significance?
04 April 2014 5,576 4 View
I heard many times in several talk that QM/MM studies can give rise to valuable approach to understand the catalytic mechanism. What if we have a crystal structure of an enzyme (which we see only...
12 December 2013 5,160 2 View
Does anyone have a very clear cut explanation for the above terms? I am unable to understand from the sources in Google. And why a detector is so important in any data reduction program ?
10 October 2013 444 3 View
I am working on enzyme crystallography and now after crystallization I want to do pH optimization of the enzyme activity. Does anybody have any suggestion as to how to get the activity by using...
10 October 2013 1,678 0 View
I am trying to model a protein structure of known sequence of unknown structure. The sequence identity of this protein is showing maximum of 54% with the template structure. When I proceed through...
07 July 2013 9,021 9 View
If a (purified) protein of an unknown gene interacts with an antibody, is it possible to get the nucleotide sequence without protein sequencing? Does any body have any explanation for this ?
06 June 2013 3,035 11 View
I am purifying a protein, initially I used to add only serine protease inhibitor (PMSF), now I want to use a protease inhibitor cocktail (from Calbiochem- 539132), where amount to be added is not...
05 May 2013 5,216 15 View
I have to do an enzyme kinetic assay using NADPH as a substrate (10 mM) and in one shot I cannot finish my kinetic assay with all the fractions collected at each step of the purification. By that...
05 May 2013 5,404 7 View
I am wanting to know whether it is important to plot standard with BSA again and again while doing the protein estimation if I am using a Bradford reagent from the same bottle (I use GeneITM kit...
05 May 2013 6,748 7 View
Can I store cell lysate in -20 degree ( the protein for structural determination) freezer for one or two days?
01 January 2013 4,184 1 View
I have purified my protein and want to estimate the amount. The plot I am getting is half of the value my senior is getting. The difference is I am using a kit (Genei TM) for Bradford's assay. But...
01 January 2013 4,285 40 View
01 January 2013 5,040 25 View
For better resolution of protein, what percentage will be better, and also for two different buffer ( for stacking and resolving buffer) only Tris-Cl of pH 6.8 and Tris-Cl of pH 8.8 is required...
01 January 2013 5,711 2 View
What is the reason for using these salts after elution? I have been using 0.1 M NaCl for purification of protein. If I use NaCl in place of KCl what may be the consequences?
07 July 2012 8,368 2 View
I am a beginner and I am using "modeller" programmer for homology modelling. and when I BLAST my query with the PDB database the first four are showing 54% identity can I proceed further? Or is...
06 June 2012 2,656 0 View
As I studied it converts a time domain into a frequency domain. What does this really mean?
06 June 2012 8,321 11 View
As I read its an expected value and the lower the value minimum represents alignment by chance. But I could not understand what the representation (for example, 1e-63 etc.) is actually denoting.
06 June 2012 6,222 43 View
My protein structure is not present in PDB and I have to compare the structure of my protein with best fit. How can I proceed? I don't even have the basics and I am quite worried about this, as...
06 June 2012 2,515 13 View
And if mutation has been done (in protein gene) and then there is no activity found with enzyme how one can monitor the function after mutation. Please explain both question if its possible.
05 May 2012 3,499 2 View
What will happen with the resultng pH if I mix a solution of pH 5 and a soution of pH 8 or any other higher pH together? Will there be any effect on mixing more than two solution with different pH?
05 May 2012 7,002 2 View
Can anybody clarify the importance and use of these values? Also I am having difficulty understanding the concepts of completeness, peptide occupancy, R-merge and Rcryst (Rfree) (%). Any...
04 April 2012 9,322 0 View
I am culturing MCF7 cell line and want to transfect the plasmid (GFP Tagged). Seniors says it is to be optimized. What is Optimation of transfection and how one can get higher transfection efficiency?
03 March 2012 2,293 10 View
Can anybody explain how to proceed with cell culture of this cell line? Thank you!
03 March 2012 828 16 View
I have to do the migration assay and I have subcultured the cell (cell count = 920000/ml) and viabilty is 96.5%. Could anyone suggest possible protocols for this as I have searched but didn't find...
03 March 2012 8,050 5 View
Please suggest. What should be the pH of this when prepared?
03 March 2012 2,615 5 View
As I have to suggest my profs.about this. Please give your opinions !! The subjects should be apart from Bioinformatics, Biophysics, and genetic-eng. enzymology and immunology.
03 March 2012 7,206 7 View
Please give a valid explanation if possible..
01 January 2012 1,843 3 View
I am working on a dehydrogenase enzyme. I wanted to check the cleavage of His-tag from my enzyme using Western Blot. When I do the Western blot in Phosphate buffer I can't even see that in the...
01 January 1970 7,860 8 View
Dear All, I am trying to express a large protein (220 kDa) with a GST tag. I performed Western Blot to check for the protein of interest. Now after using monoclonal antibody (Mouse Ab), I have...
01 January 1970 5,637 10 View
I am looking forward to design a drug against Mycobacterium tuberculosis, so what are aspects (please point out some examples) should I look for ? We can target some enzymes which are not involved...
01 January 1970 2,692 11 View
Dear all, I am trying to check the interaction between two of my proteins of interest. However before trying any biophysical technique such as SPR/ITC/MST etc, I used normal superdex-200 gel...
01 January 1970 6,866 5 View
Dear All, As we all know that lower Kd (micro-molar/nano-molar range) are considered as good interaction. But how one can appreciate the relative concept of this strength when I am measuring a...
01 January 1970 7,785 5 View
Dear all, I have been trying to look at the interaction of a protein with peptide that is 1-2 kDa. I used pull down method by streptavidin magnetic beads, as peptides are biotinylated. However,...
01 January 1970 2,423 11 View
As I read that, designing assay for cysteine protease is a bit challenging compare to other proteases, I am wondering why it is so ? Many research articles follows fluorescence based assay for...
01 January 1970 6,724 5 View
Dear All, I am trying to express and purify a protein of MW 220 kDa (full length). It has an extra GST tag in it. The strain I am using is Rossetta (so issue of rare codon is ruled out). Now,...
01 January 1970 9,000 6 View
Dear All, I am totally novice in cell culture so sorry if this question seems to be trivial to you. However, I can assume that clumping is no good for when you do grow the cells after...
01 January 1970 2,888 1 View
Dear All, I am purifying my protein (theoretical PI = 8.9) in a buffer of pH =7.1. till Ni-elution I kept the DTT concentration (1mM). Usually, after elution people used to keep DTT concentration...
01 January 1970 8,403 4 View
I have infected my insect cells (SF-21), with P4 viral titre. Now the protocol suggest that it should be harvested before lysis. Can anyone suggest me how I will figure out when to harvest after...
01 January 1970 5,578 6 View
