Avinash Sunder

10 Questions 16 Answers 0 Followers

Questions related from Avinash Sunder

Cloning without using NdeI?

I am trying to clone a gene that has a NdeI site in the sequence, so I wouldn't be able to use NdeI site in the forward primer for start codon. Apart from using NcoI, what other options are...

02 February 2018 4,754 5 View

Can anyone help with a query on enzyme inhibition and activation?

I'm studying the inhibition of my enzyme with an alternative substrate, i.e. a substrate of a homologous enzyme which my enzyme does not hydrolyze (so the compound is not a strong inhibitor). When...

08 August 2014 6,940 11 View

In enzyme kinetics does this signify substrate inhibition or product inhibition?

I am working on a tetrameric enzyme, for which the velocity of reaction increases to a maximum and then starts decreasing with higher substrate concentrations without any apparent flat region. I...

07 July 2014 5,557 9 View

Can guanidine hydrochloride cause an increase in enzyme activity?

I'm testing the effects of denaturants on my enzyme, and incubation of the enzyme with 0.5 M Gdn-HCl (4h) gives me a significant increase in enzyme activity (almost double that of control). The...

05 May 2014 8,309 2 View

How do you explain protein precipitation after ni-affinity purification?

I'm trying to purify a protein using HIS-select Ni-affinity column (washing with 25mM Tris pH7, 0.3M NaCl, 10mM imidazole and elution with the same buffer + 250mM imidazole). The protein (pI= 8.3)...

11 November 2013 5,286 6 View

How do I optimize the amount of enzyme in an assay?

How do we optimize the amount of enzyme to be used in the assay? I tried a time course for 0.5, 1, 2 and 4 uM enzyme with excess substrate, but the enzyme seems to be highly active and the...

10 October 2013 8,669 4 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...

08 August 2013 3,225 4 View

Growing high-density cultures for protein expression in E.coli?

I'm studying expression of an enzyme in BL21 star cells (cloned in pet 28b) , the protein is expressing well in the soluble fraction and it is active. But the cell mass obtained is very less in...

08 August 2013 4,334 8 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...

06 June 2013 9,825 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...

05 May 2013 9,334 10 View