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Questions related from Bhaskar Chatterjee
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 2,824 1 View
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 9,214 3 View
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 4,897 5 View
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 2,429 4 View
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 9,494 0 View
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 5,344 1 View
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 5,119 4 View
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 1,223 1 View
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 2,734 59 View
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 678 0 View
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 6,942 13 View