25 Questions 14 Answers 0 Followers
Questions related from Sumit Mukherjee
Hi I am planning to do protein N terminal amine isotope labelling analysis (N-TAILS) to find the substrate proteome of my protease of interest. For this I have the WT and the knock down line for...
10 October 2019 6,692 3 View
Hi, I am doing a pulse chase assay following radioactive methionine/ cysteine uptake. Over time I see processing of protein (67KDa to 45 KDa) following pull down with anti-HA coated magnetic...
08 August 2019 8,482 4 View
Hi, I am doing a pulse chase experiment and looking for 35-S methionine/cysteine incorporation in my protein of interest. Following gel run, I directly dry the gel (10% SDS-PAGE, 1.5mm thick,...
07 July 2019 1,277 8 View
Hi, I am working with a protein that is typically secreted from the parasitic cells that I study. I am trying to express and purify it from HEK cells. Despite of using the mammalian signal...
04 April 2019 7,501 6 View
Hi, I am doing starvation assays with Leishmania parasites for which I need to culture parasites in PBS for about 6 hrs. Typically I find that without serum supplement these cells become very...
01 January 2018 2,196 3 View
Hi, I want to make a 10% SDS-PAGE gel in which both the resolving and the stacking would contain 6M urea. How would I prepare them? I have already prepared my samples in buffer containing Tris-Cl,...
10 October 2017 8,147 2 View
Hi, I want to make a 10% SDS-PAGE gel in which both the resolving and the stacking would contain 6M urea. How would I prepare them? I have already prepared my samples in buffer containing...
10 October 2017 2,547 4 View
Hi, I want to make a 10% SDS-PAGE gel that contains 6M urea. Both the resolving and stacking buffers should have urea. Could anybody please suggest how to do this? I prepared my samples in buffer...
10 October 2017 543 2 View
Hi, I have a very general question. Since PBS is a isotonic solution, I was wondering if 2X or 0.5X PBS could be considered as hyper or hypo osmotic conditions respectively? I know that typically...
09 September 2017 2,269 3 View
Hi, I was wondering if any one could explain me the relationship between mitochondrial membrane potential and superoxide production? In order to maintain its functionality, mitochondria need to...
09 September 2017 8,811 3 View
Hi, I work with Leishmania parasites and would like to measure the free cytosolic Calcium concentration in them. For this I plan to use Fluo 4-AM dye from Thermo. As I see mostly people use some...
07 July 2017 2,709 5 View
HI, I study lipid metabolism in Leishmania parasites and have generated some lipid mutants. I would like to measure the cytosolic calcium in these mutants and compare them with that of the wild...
04 April 2017 8,117 2 View
Hi, I study lipid metabolism in Leishmania. Recently, I had some interesting observations and I would like to have your comments on these. Compared to the wild type parasites, the lipid mutants...
10 October 2016 8,638 8 View
Hi, I see often people use 5% 2-ME in the SDS PAGE sample buffer. However, mistakenly, I added it at 2.5%. Will it be a major issue? Thanks
08 August 2016 5,495 14 View
Hi, I would like to measure the changes in plasma membrane permeability for Leishmania parasites. I was wondering if there is any flow cytometry based method to do such analysis? If there is...
08 August 2016 8,720 4 View
Hi, I am working on Leishmania parasites and following treatment with some drugs, I wanted to measure the health of the mitochondria. For this, I used JC-1 dye. The drugs that I use interfere with...
07 July 2016 8,867 1 View
Hi, I am planning for an assay for which I will have to spin my samples at 110000g. We have an Beckman ultracentrifuge with a fixed angle rotor - Type 70.1Ti. On the screen of the centrifuge there...
07 July 2016 1,022 3 View
Hi, I am planning to do some luminescent assay to measure ATP production of my samples. I have some black 96 well plates with clear bottom. I was wondering if I could use them for the assay? Also,...
06 June 2016 1,610 4 View
Hi, I have a simple question to ask! What are the pitfalls of indirect immunofluorescence studies with polyclonal primary antibody aimed for determination of subcellular localization of a...
09 September 2015 8,887 1 View
HI, I am running through hard times getting my desired DNA fragments after gel purification. I want to knock out my GOI by homologous recombination in model system. For this, I cloned 5’UTR of...
07 July 2015 6,487 6 View
Hi, I have a large quantity of plasmid DNA to to digest with EcoRI and HindIII. What is the largest amount of DNA that I can digest in a single tube? Also, What should be the total reaction...
02 February 2015 4,839 4 View
Hello, I have been trying to knock down a particular protein in Jurkat cells using the Clontech's pSingle shRNA vector system. This is a doxycyclin inducible system. Initially I followed...
09 September 2014 3,497 0 View
Hi, I would like to transfect Jurkat T cells with some plasmids. So far I have used commercial reagents and every time got to see transfection efficiency nearly 70-80%. However, since these...
08 August 2014 3,497 2 View
I have been trying to incorporate two point mutations in a 8kb plasmid using the Quick Change II site directed mutagenesis from Stratagene. The two mutated sites are 8bp apart. Also, I used the...
06 June 2014 7,560 18 View
06 June 2014 792 2 View
