21 Questions 56 Answers 0 Followers
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...
15 January 2020 4,147 2 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...
02 April 2018 9,993 10 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...
01 June 2016 207 11 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...
28 May 2016 7,758 3 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...
27 May 2016 2,229 6 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...
04 May 2016 1,226 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...
24 April 2016 8,588 8 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...
19 April 2016 679 3 View
Dear all, of course, histidine is used to elute, so it should not be present in high concentrations except for the elution buffer. It is also widely known that arginine may chelate nickel and...
16 April 2016 1,587 3 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...
14 April 2016 9,847 5 View
Dear all, my protein of interest appears at 27 kDa on SDS-PAGE (Novagen Novex NuPage Gel System, denaturing, Marker: GE life sciences low molecular weight marker) - calculated molecular weight is...
10 March 2016 7,345 12 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...
05 November 2015 4,336 3 View
To test for activity of a poorly characterized cytosolic phosphatase expressed in e. coli, I am looking for a suitable buffer and substrate (p-Nitrophenylphosphate?) to run activity tests. Any...
04 September 2015 2,328 3 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...
04 June 2015 4,180 5 View
Dear all, I lately tried to establish "FastCloning" in our working group, which failed badly. The original publication can be found here: http://www.biomedcentral.com/1472-6750/11/92 I find the...
03 February 2015 8,557 7 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....
19 November 2014 8,691 1 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...
14 September 2014 5,994 2 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...
25 May 2014 5,385 13 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...
30 April 2014 4,868 1 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...
13 July 2013 1,115 5 View
I am relatively new to the field of microbiology and could need some help of more experienced scientists in the field: I have grown an overnight-culture of E.coli BL21(DE3) in LB to an (apparent)...
04 June 2013 2,060 42 View
