Avinash Sunder

11 Questions 12 Answers 0 Followers

Questions related from Avinash Sunder

How do I estimate the Km and Vmax values for two substrate (ordered) enzyme kinetics?

I am currently trying to characterize the kinetic behaviour of an oxidoreductase enzyme that requires NAD as the second substrate. I have two initial rate curves that correspond to v vs [A] at...

23 August 2018 5,718 11 View

Can anyone help refining a protein structure with bad geometry (but good R-factors)?

I'm currently refining an enzyme structure (data from X-ray crystallography). Resolution is 2.8 Å; model was generated using MR on a template with 62% sequence identity and Autobuild (Phenix). I'm...

09 November 2015 2,851 8 View

How can I find and plot the center of mass of a protein using Coot/Pymol?

I need to plot the centers of mass of different monomers of a multimeric protein and the distances and angles between them, to explain some changes in the orientation of the monomers. How can I do...

10 June 2015 4,196 2 View

How can I interpret enzyme kinetic data showing apparent cooperativity and substrate inhibition?

I've got a kinetic data (see attached fig) for my enzyme that is very confusing, and I'm neither sure what it signifies nor am I able to plot it and interpret the parameters convincingly (I'm...

10 July 2014 1,572 14 View

Are there some protein structure-based methods to support the cooperative nature of enzymes?

I am working on kinetics of a tetrameric enzyme, and a "Allosteric sigmoidal" plot on Graph Pad gives a better fit than a standard MM plot. I suspect that the enzyme shows positive cooperativity...

30 June 2014 8,098 5 View

What enzyme sample should be used for kinetics?

I'm doing kinetics studies for my enzyme. I just wanted to know which additives to use with the enzyme to determine km and vmax. My enzyme is stable in acetate buffer, but the activity is enhanced...

21 May 2014 5,500 11 View

Protein precipitation problem - where did it go wrong?

I am purifying an enzyme with C-terminal, his tag is expressed in E.coli. I get a clear pure ptn after Ni-NTA elution with 200mM imidazole. I stored the fractions at 4° C for 24 hours and then...

07 September 2013 5,670 3 View

Both recombinant and self-ligated plasmid in the same clone?

I am trying to clone my gene in pet22b (using NdeI/XhoI double digestion for 4h, gel extraction and ligation at 16 deg overnight). I got a couple of positive clones in colony PCR, when I extracted...

06 August 2013 9,194 3 View

PCR not working with similar primers

I'm encountering problems with amplifying the same gene when I use different primers - F1: GGC TAG TCT AGA ATG (TGT ACG CGG TTC GTT TAT CTG GAT C) R1: CGA GTA GAT CTC (TAG AGC CCC GCG AAT TCA AAC...

11 July 2013 2,595 3 View

Expression of periplasmic proteins

I want to clone a protein from a gram-negative bacterium that has a natural periplasmic signal peptide. Is it ok to delete the signal sequence, put the N-terminal amino acid directly next to a...

15 June 2013 9,945 1 View

How do I design primers to express a protein with C-terminal His tag?

I'm trying to clone and express a protein in E.coli using pET26b or 33b vector. I am designing primers using the NcoI and XhoI restriction enzymes. I want to know how to design primers to include...

27 May 2013 9,840 9 View