Megha Abbey

7 Questions 18 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 1,622 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 3,104 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 9,842 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 2,831 16 View

Why does the fluorescence polarization increases and then decreases in my competition assay with unlabelled peptide?

I have developed an in vitro fluorescence polarization based assay using a N-terminus labelled FITC peptide. The peptide is 21 amino acids long and the binding protein is 50 Kda in size. I am...

21 June 2017 2,434 1 View

Unlabelled peptide does not displace FITC-peptide in fluorescence polarization assay?

I am trying to develop a fluorescence polarization assay. I have optimized the assay buffer conditions. The Kd for my protein-peptide interaction is around 5uM. I tried displacing FITC-peptide...

16 January 2017 7,131 4 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 4,996 0 View