Megha Abbey

5 Questions 9 Answers 0 Followers

Questions related from Megha Abbey

On column TEV cleavage?

Hi, I have cloned my protein with TEV protease site (His tag-TEV site-linker-protein). I purify my protein with Ni-NTA and then do on column TEV cleavage (overnight). I hardly see any TEV...

11 March 2020 1,455 3 View

Cloning a large gene from cDNA?

Hi, I am trying to clone a 10Kb gene from tissue cDNA. The entire gene doesn't amplify, so I made short fragments of 2.5Kb (even 5Kb fragments cannot be obtained). The 2.5 Kb fragments have...

25 May 2019 3,064 6 View

Why Lag phase and burst phase in enzyme activity by an otherwise inactive enzyme?

Hello, I am trying to develop a fluorescent based activity assay for my enzyme. As a preliminary expt, I monitor the activity of the enzyme at different time points for increasing conc. of the...

25 January 2018 9,804 16 View

Negative shift in Tm in DSF?

Hi, I am performing DSF (Thermoflor) expt for my protein to check its binding with a ligand. The protein gives a nice transition curve without the compound. Upon titrating the compound on the...

08 July 2017 2,799 16 View

FP signal is fine but fluorescence intensity decreases upon adding protein in my FP assay?

Hi, I am new to FP assays. In the initial expt. to check binding, I am using FITC-peptide (50mM) and titrating it against 0-10uM of protein. I tried 4 conditions: a. 20mM Tris pH 7.4, 0mM NaCl b....

11 November 2016 4,967 0 View