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Questions related from Gaurav Kumar
Dear all, I am working on thermal stability of proteins. I have done CD analysis of protein samples at varying temperatures within 190-240nm wavelength and recorded the CD data as molar elliptical...
25 July 2016 4,709 14 View
I have queries related to Km, Kcat, Kcat/Km results. I have created point mutations in my proteins (beta-lactamases) and I have checked in vivo expressions. The mutant proteins seem inactive as...
09 June 2016 2,091 5 View
I am planning to study the effects of point mutations in beta-lactanmase coding genes in bacterial cells. I wish to do it manually than kit based method. I would like to know the most frequent and...
27 January 2016 4,069 8 View
I need to know the purpose of hysteretic analysis in case of enzyme kinetic studies. Thanks.
02 December 2015 6,170 2 View
A very basic query regarding the solubility, stability and storage of carbapenems antibiotics such as Meropenem trihydrate or Imipenem monohydrate. I have known to dissolve them in DMSO in higher...
06 October 2015 6,205 8 View
The research papers have variations regarding use of buffer system for the same protein in kinetics studies (Tris-HCl, HEPES or Phosphate). Is the selection based on purification buffer used for...
21 September 2015 7,173 9 View
I am trying to find out the moelcular weight of proteins using MALDI-TOF. My protein sample contains 10mM tris, 75mM NaCl pH 8. I am adding Sinapinic Acid in 1:1(v/v) ratio with protein sample. I...
15 September 2015 270 6 View
Here I have attached SDS-PAGE gel picture of Ni-NTA affinity chromatography elution profile of a protein at varying concentration of imidazole in tris buffer(pH 8). I have done washing in 10, 25...
29 August 2015 953 7 View
Can We store bacterial cell pellet (induced for protein experssion) in -20 degree for short term period? Does short term storage of pellets at low temperature has any lethal effect on expressed...
14 August 2015 5,143 4 View
I have two queries regarding the IPTG solution. First is about its storage condition in solution form? Second is about its toxicity level during induction on expression host cells (Bacteria). Is...
08 August 2015 5,488 4 View
I want to regenerate used columns to avoid experimental lags due to delay in fresh kits arrivals. I have read some articles to regenerate them but they seems a bit lengthy. Has anyone regenerated...
03 July 2015 2,679 2 View
I am cloning a gene into a pET28a vector. I used NEB enzymes NheI and HindIII to to double digest my insert and the vector using 2.1 NEB buffer. Incubation was done at 37 degree overnight (10...
29 May 2015 2,050 4 View
I am designing primers to get a soluble construct of a protein by removing the signal sequence from N-terminal region and membrance anchor region from C-terminal of the protein. I am using pET28a...
13 May 2015 1,066 6 View
What are the good servers for detecting signal peptides apart from SignalP 4.1 and Signal blast? Sometimes these servers fail to detect the presence of signal peptide in a given protein sequence....
07 May 2015 5,396 2 View
What is the major difference between a chemical competent cell and electrocompetent cell? Can we use elctrocompetent cell for heat shock transformation or vice versa? Thanks
21 April 2015 168 4 View
I have cloned a beta-lactamase gene in BL21 E.coli strain using pET28a vector for expression and in vitro studies. After Ni-NTA column purification of this His Tagged protein, I am getting two...
20 January 2015 6,299 16 View
My His-tagged purified protein contains 10mM trisCl, 300mM NaCl and 200mM imidazole at which the protein was eluted at pH 8.0. What should be ideal dialysis buffer composition to remove...
19 January 2015 7,964 9 View
I am cloning a beta-lactamase gene in pET28a vector. I have done PCR with deep vent polymerase. My next step is digestion followed by ligation in pET28a vector. What is better? PCR clean up or gel...
28 November 2014 452 8 View
I tried to prepare electro competent cells with 10% glycerol. I found it difficult to pellet them down properly. The cells adhere very loosely to the walls of tubes after centrifugation. This...
28 November 2014 2,173 4 View
I set up a PCR reaction volume of 50ul with Taq polymerase and I found distinct bands of amplicon but when I double the reaction volume to 100ul to get higher volume of amplicon I found multiple...
23 November 2014 2,823 16 View
I am studying in vivo expression using pBAD18 Cm vector in BL21 cell strain. I am confused about the induction procedure. I am using 0.2% arabinose to induce pBAD promoter at 0.2 OD growth. Can I...
12 November 2014 8,558 10 View
I get multiple protein bands in SDS PAGE after expression in BL21 cells. Does sonication results in protein degradation and result in extra overexpressed bands? I sonicate for 30 seconds with a...
03 November 2014 9,903 12 View
Can periplasmic proteins such class A beta-lactamase be secreted outside gram negative cell? The protein is supposed to be anchored in periplasm through inner membrane. Can anyone has right...
10 October 2014 475 2 View
See above
08 September 2014 2,431 4 View
What is the best composition of lysis buffer for E.coli cells carrying recombinant proteins? How can we increase the efficiency of lysis buffer to enhance downstream processing of the proteins?
23 August 2014 1,767 5 View
I have read about the fragile nature of transformed BL21 cells for expression studies. I have been using the transformed BL21 cells stored at normal refrigerator temperature for expression...
25 July 2014 3,199 5 View
My plasmid construct has two shine-dalgarno sequence. One is present 55bp upstream of gene and other is present in my primer. Does the presence of two SD affect the expression of my gene?
23 July 2014 3,976 4 View
I want to do knock out of SHV class of beta-lactamase from Klebsiella pneumoniae genome as a part of my PhD work. I am confused with the techniques available. I have never done knockout before....
19 July 2014 3,072 2 View
