Bhaskar Chatterjee

11 Questions 20 Answers 0 Followers

Questions related from Bhaskar Chatterjee

Why mP values in a FP assay are decreasing?

I initially did an FP assay with receptor (A) and a flourescent labelled ATP analog (B). For the first three times, i tried varying B conc for a fixed A concentration. I found increase in mP...

04 April 2019 2,826 1 View

Co-transfection of 2 plasmids in HEK-293 cells

I am trying to co-transfect 2 plasmids in HEK-293 cells. One is pcDNA-3 based( protein A) and the other is pcDNA5/FRT/TO based GFP-tagged ( protein B). Protein A's expression is optimized. Protein...

07 July 2017 9,216 3 View

Anyone knows the IC50 values of Geldanamycin in HeLa cell line?

I am doing MTT assay on Hela cells. i keep the drug for 24 hours after cells have attached and become confluent in the 96 well format. After that i add MTT at 0.75mg/ml and incubate for 2.5 - 3...

04 April 2016 4,899 5 View

Does heat shock to Hela cells produce certain chemicals or changes in cell morphology that affect the MTT assay?

After a certain incubation period at high temperature, I am getting higher MTT readings as compared to smaller incubation periods at the same temperature. Cell viability should ideally decrease...

05 May 2015 2,432 4 View

Which cell lines shuld i use for my experiments?

The experiment i have decided to conduct is as follows: I want to overexpress luciferase in certain cancer cell - lines. After transient transfection, i then intend to overexpress Hsp90 in those...

02 February 2015 9,496 0 View

Is ATP regenerating system mandatory to use when measuring ATPase activity of a protein?

I am using a pretty old ATPase protocol that measures inorganic phosphate released by The Fiske and Subbarow method( 1925). Nothing is mentioned about ATP regenerationin the protocol. Should I add...

02 February 2015 5,346 1 View

Can inhibitors function independent of concentration.?

I am doing aggregation prevention assay of one substrate and a chaperone. the chaperone prevents aggregation upto 90% at 1:10 molar ratio. Now I add a certain inhibitor of the chaperone to see how...

10 October 2014 5,122 4 View

Use of Geldamaycin at 500nm for monitoring aggregation studies using substrate and chaperone?

Hi, i am looking to use Hsp90 Inhibitor Geldanamycin. I have optimized the assay where i have a substrate protein and this chaperone and i have monitored the prevention of aggregation using a...

02 February 2014 1,225 1 View

Why am I getting two light bands on SDS page along with my protein of interest?

I am trying to purify mammalian HSP90 from E.coli Bl21DE3 cells. They are histagged. I use Ni-NTA, followed by Anion Exchange chromatography and then GFC in Sephadex 10/300. My protein has a mol...

01 January 2014 2,735 59 View

Which is the best buffer for long term storage of proteins at -80deg C?

Right now my protein is in phosphate buffer. Phosphate buffers have a huge jump in pH upon freezing so I need to store it in a different buffer. I'm not ready to add glycerol for -20 as when I...

06 June 2013 679 0 View

Should I remove imidazole from protein samples before using native PAGE?

For running my samples in native PAGE, should I remove imidazole by dialysis? I have done Ni-NTA followed by size exclusion chromatography and then concentrated the samples using Amicon Filter...

04 April 2013 6,952 13 View