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Questions related from Peter Rehbein
We are repeatedly troubled with strange Protein bands in PAGE after PCR using Q5 polymerase from NEB. Who has measured the molecular weight of Q5 already and could help us out? Thank you all in...
01 January 2020 6,011 3 View
I am writing a script for Processing fluorescence spectra. In Order to make it useful for a large audience, I am collecting Sample Data of Export Files from fluorescence spectrometers of different...
04 April 2018 8,069 11 View
Dear all,I was able to identify a new, synthetic molecule which is able to induce expression in bacterial gene regulation (only a particular operon is effected) - The practical use of this...
12 December 2016 7,182 0 View
Dear all, after IMAC elution, my E. coli expressed protein (pI=6,32, MW=13 kDa) is in 400 mM Imidazol and 0,3 M KCl. TEV-Protease and cleaved off His-Tag are also swimming in this solution. I want...
06 June 2016 3,873 12 View
Dear all, I am using ammonium sulfate precipitation of protein as a first purification step from my crude bacterial lysate.The method works well, the proteins precipitate in sequential order as...
05 May 2016 3,867 9 View
Dear all, lately I get some serious contamination in my E. coli cultures. I suppose it is a fungus of some kind. I am using minimal medum with no trace elements and no vitamins, the only organic...
05 May 2016 9,043 46 View
Dear all,I am trying to purify recombinant prion protein (PrP).The chromatogram during gel permeation chromatography shows that nearly half of my protein exists in the fom of dimers. Running...
05 May 2016 10,027 4 View
Dear all, my latest gel permeation purification produced some strange results (see chromatogram attached).I am purifying a small 14 kDa protein after Ni-NTA-affinity chromatography and subsequent...
05 May 2016 1,179 7 View
Dear all, my E. coli lysate is extremely viscous, to the point that it is impossible to apply it to the column for purification. What can I do?I added 500 units of DNAse to 50 ml, but the...
05 May 2016 9,709 8 View
Dear all, I am planning on using a mixed bed resin (ion exchange bead material) for deionization of my 8 M urea stock solution.I need this solution at pH 2 - will the ion exchanger have effects on...
04 April 2016 1,301 9 View
Dear all, I wondered which amino-sugars can be digested by E. coli.Sure, Glucosamine can be metabolized by these bugs. But which other amino-derivatized carbohydrates can they devour?What about...
04 April 2016 4,434 4 View
Dear all, we all use prepacked chromatography columns for affinity binding in protein purification (most prominent metal affinity like IMAC). I always wondered whether it would be better to load...
04 April 2016 602 5 View
Dear all, I noticed that the pH rises when I dilute a 2 M stock solution of imidazole (pH 8.0) into a 0,2 M phosphate buffer (pH 8.0) to 500 mM imidazole (His-Column elution buffer). The buffer...
04 April 2016 10,070 3 View
Dear all, I am trying to get acquainted with "CCPN", a free NMR analysis package.The software looks very nice and offers many features, however, it produces error after error when I am trying to...
03 March 2016 6,814 4 View
Dear all,I add sucrose to my lysis buffer (E. coli culture) in order to prevent aggregation of my target protein during lysis. When I centrifuge the lysis mixture (13000 g for 15 minutes), I get...
01 January 2016 8,392 6 View
Dear all, I am currently designing a lysis buffer for my protein of interest, expressed in E. coli BL21(DE3).The protein's solubility crucially depends on the correct formation of its single...
11 November 2015 2,014 3 View
Dear all, I think that BL21(DE3) is crippled in the periplasmic oxidating route, but is it really completely dsbC and dsbA deficient? If so, this would mean that it is not even possible to have...
09 September 2015 9,192 5 View
Dear all, in a recent experiment on auto-induction, I included a control with only lactose as carbon-source for BL21(DE3). I expected the Colis to grow on this sugar, but the solution stayed...
08 August 2015 9,812 8 View
Dear all, I am trying to elute my protein of interest (POI) from Ni-NTA column without using imidazole, since this is incompatible with Cation-Exchange-Chromatography, which will be the subsequent...
06 June 2015 982 5 View
Dear all,I am trying to express my protein of interest (aggregation prone) in a soluble form and decided to give a try to a periplasmic leader sequence. I was hoping for disulfides to form...
05 May 2015 9,449 10 View
Dear all, I am playing around with some new methods for bacterial expression and I want to use a model protein for which expression optimization will make the most sense.I looked at somatotropin...
03 March 2015 9,887 3 View
I understand that proteins with disulfides needed for biological activity/correct fold need to be expressed in the periplasm. I wondered whether proteins without disulfides (cytosolic proteins)...
02 February 2015 8,254 3 View
Dear all, for a synthesis, we need 4'-OH-Monosaccharides (all other hydroxyl-groups should be protected, acetylated or benzylated or else...) - I am desperately looking for a working protocol to...
12 December 2014 4,716 8 View
Dear all, For a synthesis, we need to mix dimethylsulfoxide with toluene, miscibility tables tell us that it should be possible, but we observe phase separation even at DMSO concentrations as low...
12 December 2014 3,067 23 View
Hi there,for a continuous enzymatic reaction (membrane reactor with immobilized enzymes), I am looking for a buffering substance that is not or only slowly degraded by thermic or other processes....
11 November 2014 4,532 1 View
I need this for a review: What is the most popular or established method for preparation of samples for electron microscopy if a sample is hazardous for humans, e.g. when forming aerosols or is...
10 October 2014 2,181 5 View
I understand that the stainability of a protein with coomassie depends on the amino acid composition of the protein - how pronounced is this effect and is there a way to somehow introduce a...
09 September 2014 5,644 2 View
Before I start cloning, I was just wondered whether somebody would be ready to share the plasmid? Thanks in advance,Regards,Peter
09 September 2014 1,041 0 View
I need to quantify expression from SDS-PAGE-gels and I am not quite sure whether I am doing it right... (I have attached a typical gel in greyscale: lane 1 Marker, lanes 2-5 standard...
05 May 2014 2,029 10 View
When preparing stock solutions for serial dilutions for determination of molar absorbance coefficients, how can I ensure that the substance is transferred quantitatively, so that I don't introduce...
04 April 2014 9,225 1 View
I performed NTA-column chromatography (Hi-Trap-His-Column, 5 ml) with loading under denaturing conditions and did on-column refolding. I elute with 800 mM imidazole and cleave the tag with...
02 February 2014 1,433 5 View
My question addresses whether the genetic restructuring imposed by the immortalization method changes the properties of the cell in any way (sensitivity for external effectors, protein expression,...
02 February 2014 8,608 0 View
I want to label my protein with Carbon-13 and understand that 13C-Glucose or 13C-glycerol is quite expensive. Are there alternatives?
02 February 2014 1,798 3 View
I am using 15N-labelled Phthalimide as internal standard for nuclear magnetic resonance measurements (Pht-Signal should give a straight line at constant shim). However, its poor solubility at pH 2...
11 November 2013 466 7 View
see above
10 October 2013 8,580 2 View
I know there is FOLDit and folding@home, but these only allow for working with structures provided by the authors. Is there a program which I can load my own pdb file into and play around with the...
07 July 2013 1,993 6 View
I am trying to grow E coli to high cell densities in order to enhance the yield of my recombinant protein. I understand that auto-induction media with appropriate aeration give good results here....
08 August 2012 9,486 1 View
I'm working on a protein that is potentially harmful when it may form aerosols. Whenever I manipulate the sample openly, I'm therefore working under a flow bench (S2-Lab). I'm also directly...
07 July 2012 2,978 1 View
07 July 2012 6,310 7 View
