Manjula Ramu

11 Questions 18 Answers 0 Followers

Questions related from Manjula Ramu

How to check quantitatively the tryptophan oxidation of a protein?

Hi everyone, I am trying test the oxidation level of a protein in presence and absence of a ligand. My protein is prone for oxidation at the same tryptophan in-vivo. I am looking for small...

10 October 2017 2,177 2 View

What is the best method to purify basic proteins with pi 7.8?

Hi all, I am purifying a basic protein with pi 7.8. I used 50mM tris 8.8, 300 mm NaCl, 5% glycerol, 0.1mM PMSFand 5mM beta ME in the buffer and performed affinity purification. Eluted with 200mM...

04 April 2015 1,973 6 View

Are co-expressed proteins always seen as 2 bands on SDS-PAGE?

Hi all, I am co-expressing 2 proteins (cloned to pETDUET1) in E.coli to check their interaction. I did the induction and checked cell lysate on SDS-PAGE. Surprisingly I observed a induced protein...

04 April 2015 8,633 7 View

How does the heparin column work for DNA bound proteins?

Hi guys, My protein is nuclear human protein and I am purifying recombinant protein from bacterial culture. when I performed ion exchange chromatography, my recovery was nil. thinking it is bound...

03 March 2015 4,433 9 View

How can I increase the binding affinity of GST-tagged protein to GST sepharose column?

Hi, I am purifying a protein having GST tag. I am using 20mM tris pH 7.5, 200mM NaCl, 5mMDTT and 5% glycerol as the equlibration buffer. Column is of 2ml bed volume (I have hardly 3mg of my...

03 March 2015 5,358 5 View

Why is the zero'th time deacetylation reaction showing higher florescence intensity than 1hour reaction?

I am trying to standardize de-acetylation assay for my enzyme and I have taken no enzyme reaction as blank. My substrate is labelled with AMC and using NAD as cofactor. I am not getting saturation...

01 January 2015 1,952 1 View

Why is my protein acting differently at the same pH?

I am isolating a protein in soluble form from Bacterial systems. I have done cation ion exchange chromatography(Q column) at the pH 8.2 using Tris Cl and HEPES buffer. Protein is binding to the...

10 October 2014 9,271 10 View

Why is my protein getting precipitated, even at low concentrations?

I am purifying a recombinant protein (host Rosetta) with his tag. I am getting reasonably good yield (4-5 mg/liter of culture). However after affinity purification using Ni-NTA column, during...

06 June 2014 6,288 5 View

Can anyone help me figure out why I am losing protein during ion exchange chromatography?

I am purifying a protein from a bacterial system and I was able to successfully purify the protein through an affinity column. My protein has a pI of 5.9 and it is stable at higher pH. After...

06 June 2014 5,925 6 View

How much of Dnase can be added and what buffer can be used for cell lysis during protein extraction?

I am isolating protein from more volume of culture. However I am unaware how much dnase to use and also what buffer to use for its activity, if I am dissolving the pellet in 30ml of lysis buffer...

07 July 2013 4,230 5 View

More proteins are going to inclusion bodies, so how does one get a soluble protein?

I am trying to express a recombinant protein in E.coli, however the problem is with its solubility. I am inducing (0.5mM IPTG) the cells at 0.7 OD and incubating at 15 degree temperature. Most...

06 June 2013 5,542 14 View