39 Questions 53 Answers 0 Followers
Questions related from Abir Chakraborty
Hi all, I am planning to perform Gelatin Zymography using conditioned media from three different knock out from same cell line. I seeded equal number of cells and collected the conditioned media...
06 November 2024 5,453 1 View
Hi All, I am planning to purify a His-tagged protein from HEK293T cells after overexpression. Could you please share your opinion regarding the right composition of the lysis buffer? I will be...
08 June 2023 4,915 0 View
Hi All, Can I use 0.2% Gelatine coated, glutaraldehyde cross-linked glass coverslips as an alternative to Poly-L- lysine coated coverslips?
07 November 2020 2,403 1 View
Hi All, Recently, I purified 8 proteins and stored at -20 with the elution buffer containing 20 mM Tris, 250 mM NaCl and 200 mM Imidazole. I autoclaved the buffer, so I do not know whether the...
26 September 2020 10,222 6 View
Hi All, We recently used HisPur-NiNTA resin for protein purification. The protocol from Thermo says that 20 mM MES,100 mM NaCl, pH 5 is required to regenerate beads without affecting Ni content....
15 August 2020 4,408 3 View
Hi All, I have some compounds and proteins for ITC. IC50 of those compounds have 0.3-0.6 uM range, depending on different cell lines. What should be my starting cell and syringe concentration in...
06 August 2020 8,209 5 View
Hi All, I recently did the thermal stability shift assay with my 30 kDa monomeric protein with different concentrations. I have noticed that it gives us three Tm as you increase the concentration....
11 October 2019 9,026 0 View
Hi All, I have some questions on the thermal stability shift assay. 1. what is the ideal hold time for thermal scanning of protein? higher molecular weight needs more holding time whereas lower...
06 October 2019 6,078 3 View
Hi all, I am planning to PCR a 7430 bp fragment from a big plasmid(11 kb) with Thermo Phusion hot start II DNA pol. Thermo recommend to use 15s-30s as extension time per 1 kb length. Overall GC%...
09 August 2019 7,597 3 View
Hi All, Can we use potassium phosphate tribasic instead of dibasic for making terrific broth? Also Can we replace it with sodium phosphate ? Can sodium phosphate buffer be used for terrific...
24 July 2019 2,120 0 View
Hello All, I just want to know your thoughts on NEB Q5 site directed Mutagenesis kit. Has anyone used this ? Is it good ? I have a plasmid of 6 kb on which I design my SDM . Your advice and...
19 June 2019 1,762 7 View
Dear All, Can we use Tween 20 instead of triton X in 0.1% PBT-T(I assume T stands for triton X only) for immunofluorescence ? Also, can tween 20 be replaced by tryton in IP wash buffer?
29 May 2019 6,349 3 View
Hi All, I am planning to clone one of my inserts into VC155 BiFC plasmid. My insert size is 8 kb where vector size is 5kb. Is it very hard to clone 8 kb into 5 kb? Also I would like to do...
22 May 2019 7,198 3 View
Hi all, I recently performed BiFC experiment to confirm the direct protein protein interaction between two protein. However, I also tried to validate specific interaction through western blot with...
04 May 2019 7,487 0 View
Dear all, I recently used Cluspro server to dock my proteins and pymol interface residues script for a prediction of amino acids involved in the complex. Now, I want to mutate few amino acids and...
24 March 2019 4,114 3 View
Hi All, I need to wash and recharge Ni-NTA column. Unfortunately, I do not have GuHCL now. Can we use 8M Urea instead of GuHCL? Is it also necessary to wash recharged beads with Urea+ 0.2 M...
07 March 2019 8,508 3 View
Dear All, My protein has a heparin binding site,and I am planning to pull down the protein complex to see whether my target protein is binding to that protein or not.I have one heparin agarose...
28 February 2019 993 2 View
Hi All, I recently generated few constructs in pcDNA 3.1 HisC,encoding three different Fibronectin fragments.I did not realised that His tag was at the N terminal(I just overlooked). I...
17 November 2018 5,307 1 View
Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o after regeneration?
01 November 2018 4,549 3 View
Dear All, This is going to be a very silly question to ask. I have digested my vector(~4000bp) with ApaI and I need to dephosphorylate to prevent self ligation. I looked at the NEB protocol for...
09 October 2018 5,198 2 View
Dear All, Does anyone have the recipe for making mineral salt medium ? I have found some information but i have few question. Mineral salt medium (MSM) had the following composition (per...
05 September 2018 407 0 View
Hi All, Label transfer technology is very popular to investigate protein protein interaction. Can this technique be used to investigate to find out the binding interface of two proteins, more...
30 August 2018 4,270 5 View
Dear All, Is there any one in this group who has successfully transfected and expressed recombinant Fibronectin fragment into HeLa or HEK293 cell.
09 August 2018 4,673 1 View
Dear all, I have been trying to express a recombinant protein which is almost 86 kDa in Pichia pastoris. My transformation into pichia genome was successful ,as confirmed by colony PCR, but I am...
08 July 2018 5,231 3 View
Hi All, I have recently done some electroporation into GS115 strain with pPIC9 containing a 2230 bp fragments. After transformation I performed colony PCR with gDNA from Pichia and used plasmid...
04 July 2018 5,965 3 View
Dear All, Can I autoclave glucose with yeast, peptone and agar to make YPD plates for Pichia pastoris? I read that 10 min sterilisation in liquid cycle would not be a problem for YPD plates. I...
11 June 2018 1,112 4 View
Hi All, I have a very basic question to ask. We generally inoculate a single colony in small culture for overnight growth following transferring them into big conical and wait for the OD 600 to...
30 May 2018 8,048 2 View
Dear All, I have a ~2300 bp insert with Snabl and NotI respectively( 5 prime to 3 prime). I need to use pPIC9 expression vector which is 8 kb in size. I co digested pPIC9 with SnabI and NotI,...
28 May 2018 3,812 3 View
Dear All, I have been doing the expression and Purification of GST-HSP90M domain in BL21 9DE30 strain. I used to use 2x YT broth, 16 degree C induction with 0.2 mM IPTG for overnight induction....
19 March 2018 1,913 0 View
Hi all, I need to take two plasmids with me to Scotland for my research work. This question seems very stupid in this group. If I add 10 ul of a plasmid on a filter paper, dry them at RT and...
03 March 2018 7,561 4 View
Hi all, I had to make some endo-tox free plasmid but there is still some ethanol in the final eluted volume.I added 80 ul TE water for a total eluted volume of 200 ul.I stored them at -20 but it...
31 January 2018 2,691 2 View
Hi all, I just wanted to know the time required for transformed pichia to grow on MD plates til the colony is visible. I have transformed with my gene of interest into to Pichia pastoris(1*109...
07 December 2017 3,649 5 View
I am planning to use Pichia pastosis for protein expression and purification. I Shall be using pPIC9 which has a secretion signal and the protein can easily be purified from the supernatant. Can...
28 October 2017 5,698 2 View
Dear all, Recently, I have been struggling to purify a protein which has 10 disulfide bonds. I have found out that the proteins are inside the inclusion bodies. I have used sarcosyl to solubilize...
18 September 2017 1,386 12 View
Dear All, This question might be stupid to all who are expert in co transformation. I tried to co transform p-RARE and pQE80l containing my Gene of Interest into XL1 blue. I used 30 microgram/ml...
07 August 2017 3,655 0 View
Dear All, I have been getting a problem during gel DNA recovery. When I checked the concentration of the DNA, purified from Gel, in the Nano drop, it is giving me 27 ng/uL, but the peak is showing...
26 July 2017 5,583 3 View
What Should I do now? I used pet vector and BL21 (DE3) strain.
30 January 2017 6,012 4 View
Hi All, I have recently used Cluspro to dock two of my proteins. I received my docked file and best model score model was named as Model000.000 something. Now, I would like to use this complex to...
01 January 1970 4,665 4 View
Dear All, It would be very helpful if some of you who are PI in Universities and Research institute and receive a bunch of postdoc application every month, share your opinion and suggestion with...
01 January 1970 9,924 5 View
