24 Questions 16 Answers 0 Followers
Questions related from Haaris Ahsan Safdari
Hi, I am trying to express a gene which belong to classical TA (Toxin-Antitoxin) module. Specifically, I am trying to express the toxin gene using pBAD vector in BL21 cells. Despite trying out...
09 March 2022 8,358 2 View
Hi, I am planning to tag a gene endogenously (in vivo) in E. coli for immunoprecipitation. Can anyone suggest an elegant protocol for this? Thanks!
30 June 2021 7,917 1 View
Hi, Can anyone suggest the optimum level of magnesium ion intracellular concentration in E.coli? Thanks!
21 June 2021 577 0 View
Hi, I am traying to optimize the expression level of my protein cloned in pBAD vector. Currently, I am inducing it at 0.002 w/v but see a significant overexpression of my protein. The current...
27 May 2021 6,367 2 View
Hi, Does anyone know of any plasmid where I can clone two genes under two promoters? Basically, I want to coexpress these protein together but with Arabinose inducible system. pETDuet,...
08 May 2020 8,361 0 View
Hi, Does anyone have first hand experience of using pETDuet-1 vector and pRSFDuet-1 vector. Both of them have two promoter followed by two MCS. Does both of them offer similar level of expression...
20 December 2019 2,814 0 View
Hi, Is there any way to prove that a protein is membrane anchored/peripheral membrane protein? Any simple trick or any technique will be helpful. Thanks
25 November 2019 1,191 5 View
Hi, Can someone please suggest which detergents to use in regular lysis buffer/resuspension buffer for E. coli cells? Please also suggest the optimum concentration to be used. Thanks.
25 November 2019 1,521 5 View
Hi all, I have a protein with conserved Threonine and one Lysine in the middle of the protein sequence. Being a charged and conserved amino acid across family domain of this protein, I have strong...
22 March 2019 3,227 4 View
Hi, I have cloned some three different constructs which are putative membrane protein of Mycobacterium tuberculosis into pET28a with N-term His tag. I tried to express them in BL21, Rosetta, C41,...
18 December 2018 8,681 2 View
Hi, I have a 1.7kb gene cloned in pET28b which has N terminal His tag. Total amino acid encoded by the 1.7 kb gene is 573. I want to mutate 398th amino acid from Glutamic acid (E) to Glutamine...
02 December 2018 7,064 4 View
Hi, Can anyone please highlight the difference between normal agarose and agarose low EEO? Any specification as to which to use for agarose gel electrophoresis? Is it that extraction of pcr band...
01 December 2018 6,835 3 View
Hi, I wanted to perform docking through SwissDock. They ask for target structure in pdb format. But the ligand should be in MOL2 format. Does anyone know how can I get MOL2 format or any...
07 November 2018 7,746 7 View
Hi, One of my colleague put the Superdex 200 10/300 GL column mistakenly in 1M NaOH at flowrate of 0.2ml/min overnight instead of water wash. Will the column be stable? How to know that the...
19 August 2018 8,259 2 View
Hi, One of my protein is 100kDa on SDS PAGE gel. I have a strong hunch that it forms trimer in the native conditions. It is eluting at correct trimer position in Sephadex 200 10/300 GL column. I...
04 August 2018 8,038 3 View
Hi, I have rosetta (DE3) with incorporation of tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. I cloned a gene in kanamycin resistant...
31 July 2018 6,688 2 View
Hi, If I express membrane protein of Mycobacterium in E.Coli cells, is it necessary to have signal peptide ? How does E. coli machinery sends it to membrane? Is there is any specific pathway like...
23 July 2018 6,094 2 View
Hi I have cloned a 44.1 kDa protein (which exist as trimer in native state) in pET28b. It is expressing well in BL-21, Rosetta(DE3) as well as C41 strain. I have attached the visualization tool...
03 May 2018 4,840 5 View
Hi, I feel that my Supherose 6 column is contaminated with some protein because I get pretty clean SDS gel bands after Ni NTA but after SEC I see multiple bands on SDS PAGE. How should I clean...
11 April 2018 1,348 3 View
hi, I am working on a membrane protein of 153 kDa mass. I eluted it with 300mM imidazole by Ni NTA. Does the centrifugal filter (100kDa cutoff) which I am using to concentrate my protein will...
07 April 2018 4,892 5 View
Hi, To concentrate a protein, what speed should be used in centrifugal filter? Does protein stick to membrane? Some people say that concentrating your protein will lead to aggregation, is it true??
09 March 2018 5,179 1 View
Hi, I am using rosetta Ecoli cells for expression of recombinant cells. After optimal time expression, I am keeping the cell pellet at -80 degree celsius. I am resuspending cells in lysis buffer...
09 March 2018 409 4 View
Hi, I am purifying a membrane protein of 54Kda (with his tag) using DDM Detergent. After Ni NTA chromatography, I can see my protein band but is mostly accompanied by 42 kDa and around 74 kDa...
08 March 2018 7,060 4 View
Hi, I am cloning a 2.8 kb gene into pet28b vector. I did two ligation reaction. In one of them, I did not put insert (only vector and ligase) (control reaction). Then I transformed 10 microlitre...
16 February 2018 1,938 4 View
