42 Questions 60 Answers 0 Followers
Questions related from Prem Prakash
I am planning to purify the mononucleosome for cryoEM study. I have seen, people assemble the nucleosome from recombinantly expressed histones and then providing DNA sequence to it. Isn't this...
20 May 2024 7,132 1 View
I have a recipe of loading dye (10 mM EDTA, 1X TBE, pinch of Xylene cynol and Orange G dye which is made upto 10 ml with 100% Formamide) but somehow the bands of the nucleotide product...
18 April 2024 7,988 6 View
Dear all, I have been trying to knockdown using siRNA (duplex) targeted against my protein of interest (# 2 lane shown in the image) and a random non-target siRNA ((# 1 lane shown in the image)...
30 September 2023 748 2 View
Looking for a grant that would provide grant in USA if any such relevant agencies available. Thank you in advance With kind regards
04 October 2022 6,052 0 View
Two different proteins which are well known for forming a hexamer ring by making electrostatic interaction among its heterodimers, reported in yeast and the structure is also available in PDB. I...
18 July 2022 3,674 5 View
Dear all, I have started performing an experiment to determine the Kd value of an exonuclease enzyme using 5'-FAM labelled Oligo substrates using Fluorescence polarization. As this enzyme uses...
23 July 2021 1,592 16 View
It is very much confusing when some papers use this term "apparent Km of an enzyme even if they use single substrate ? Not at all clear ! Please help me understanding this. Thanks in advance.
28 September 2020 3,760 7 View
I am working on an enzyme, which Km I have calculated after its rate saturation. I have solved the crystal structure of this enzyme and wanted to perform a mutation in the active site based on its...
23 November 2017 7,690 9 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...
27 September 2017 1,459 4 View
I am separating the hydrophobic component from the cereals, and I am concern about the solvent used to such process. Is there any solvent (other than ethanol and PEGs) which has no hazardous...
30 December 2015 7,328 1 View
I have done the flourescence spectrometry experiment with my protein and ligand. Keeping the protein concentration constant with increasing concentration of the ligand (here NADH). I saw there is...
04 June 2015 8,244 4 View
I have done site directed mutagensis by designing the primers at the mutation site, and whole plasmid amplification was performed using Hifidelity enzyme XT-20 (which can amplify up to 20 kb). The...
17 December 2014 952 12 View
I want to validate my protein structure, is it safe to use these online servers?
28 July 2014 8,919 7 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...
20 May 2014 4,432 9 View
I am confused, I am building a molecule and one refinement was done the gap of R free and Rmeas is 3%, so to reduce this huge gap if I join the broken chains having lesser density or side chains...
13 December 2013 8,689 6 View
Why it is problematic during molecular replacement or any other simulation, is there any comment ?
01 December 2013 8,693 8 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 ?
09 October 2013 9,044 3 View
After an X-ray diffraction takes place some spots appear as two dimensional called refection? I am curious to know what do they actually represent? Is it an atom, group of atoms, a whole protein...
28 August 2013 2,024 5 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 ?
09 June 2013 7,409 10 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 3,209 6 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...
23 January 2013 7,647 24 View
Can I store cell lysate in -20 degree ( the protein for structural determination) freezer for one or two days?
02 January 2013 5,135 1 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.
14 May 2012 5,620 2 View
Please suggest. What should be the pH of this when prepared?
18 March 2012 7,105 5 View
Can anybody explain how to proceed with cell culture of this cell line? Thank you!
14 March 2012 471 15 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.
07 March 2012 2,273 6 View
Dear all, Apologies for an off topic. I am curious to know if someone wants to pursue a second PhD in order to switch into a closely related field from his/her first PhD, would that be considered...
01 January 1970 8,984 4 View
Dear all, Greetings ! I am a total newbie in enzymology as I started working on this very field couple of months ago. I am working on a nuclease which degrades oligomers of nucleotide from 3'...
01 January 1970 204 17 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 5,729 6 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 4,933 3 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 8,438 4 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 5,016 9 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 1,617 5 View
Dear All, My initial or original predicted model of protein has all the amino acid sequences in the polypeptide chain. But when I docked this structure with its partner protein, the resultant...
01 January 1970 2,171 9 View
Dear all, I got results after performing an enzyme kinetics with 4 different substrate the kcat of B and D is respectively two times lower than A and C. However, I am seeing the higher Km of A...
01 January 1970 8,755 4 View
Dear all, I am looking for a commercially available pull down assay kit that has conducive buffer conditions and high affinity for the antibody conjugation. My protein of interest (bait) shows a...
01 January 1970 3,501 5 View
I am performing pull down experiment followed by western blot. Two interacting partners are involved in the experiment, I need to pull down one of them. How much protein should I use for interaction.
01 January 1970 299 1 View
Dear all, I have been trying to knockdown my target protein using siRNA. The protein has several isoforms the effect of this knockdown has been showing up in the functional assay. However, I...
01 January 1970 9,408 7 View
Dear all, please suggest or provide a link for the provider or company who can design or synthesize shRNA plasmid (Single vector system) that will directly express into the mammalian cells to...
01 January 1970 8,936 1 View
My target protein is a membrane protein and I want to check its expression level in the test sample using western blot. Can anyone suggest which protein should be used for loading control, like we...
01 January 1970 2,688 5 View
Dear Experts, I am planning to perform an EMSA to test a protein-protein interaction. Could you please suggest some fluorophore labeling which is not large protein like GFP (as we want to avoid...
01 January 1970 6,831 3 View
Dear all, I want to express my protein of intterest in absence of salt. Now the challenge is to see the survival of cells and even if it survives, where I could find a cell medium (any company...
01 January 1970 7,080 5 View
