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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...
15 January 2020 4,206 2 View
Dear all, recently, I observed by fluorescence microscopy that a recombinant Antibody Fragment opted for secretion amasses in the late(?) Golgi. Producer cell Line is CHO Derivative K1. What may...
31 August 2018 707 0 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 10,058 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 273 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 7,811 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 2,286 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 1,278 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 8,654 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 733 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 1,638 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 9,916 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 7,399 12 View
Dear all, due to a couple of rare codons in the sequence of my protein of interest, I am using BL21 Codon Plus RP for expression from a pRSET A vector. Because the pRSETA conveys ampicillin...
05 March 2016 4,990 10 View
Dear all, would it make sense to include Triton X-100 in the washing step during Ni-affinity chromatography of E.coli lysate in order to clear out endotoxins?Normally this procedure is only...
23 February 2016 5,433 4 View
Dear all,which would be the most straightforward NMR-experiment to check on the cis/trans state of the prolines in a protein ensemble?Thanks for any help,Best regards,Peter
15 February 2016 1,688 3 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 4,372 3 View
I am trying to gather information on the mechanical properties (force measurements, stability until breakage etc.) of silica coated biopolymer beads - I did a literature search, but there is very...
24 September 2015 7,868 2 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 2,391 3 View
Dear all, I am trying to reproduce results from this paper:http://www.ncbi.nlm.nih.gov/pubmed/16233057When I try to express the construct (PhoC - I cloned in a different vector, pRSET A) the...
06 July 2015 1,102 5 View
Dear all,I have the suspicion that my protein is aggregation prone - what would be a simple assay to test this? I understand there is the Congo Red Assay... Could anyone provide details...
05 June 2015 6,001 9 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 4,235 5 View
I know that there is the kazusa database, but the entries there are only listing entries related to the codon usage in the COMPLETE GENOME. Are there databases existing for more specialized codon...
08 March 2015 9,423 1 View
Dear all, I want to express my protein in the periplasm of E. coli and decided to use the common pelB-leader-sequence for translocation to the periplasmic space. Now I also want to experiment with...
09 February 2015 3,837 4 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 8,617 7 View
Dear all, are there known fusion partners or amino-acid-subsequences that provoke precipitation of the fusion protein in inclusion bodies?I want to provoke inclusion body formation, caused solely...
24 January 2015 3,397 5 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 8,744 1 View
Dear all, I am facing an issue in molecular cloning - I have a cloning vector and an expression vector and want to replace a certain sequence from the expression vector with an insert stemming...
12 November 2014 6,670 9 View
Dimers should have the same isoelectric point as the monomer and should therefore not be separable from the monomer, or is this not correct?
01 November 2014 2,496 14 View
I want to purify a 20 kDa Protein from an eluate (polishing after affinity chromatography, His-tag purification) and want to use size exclusion chromatography for this. I need to pack the...
01 November 2014 7,579 4 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 6,046 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 5,445 13 View
I understand that the effect of glucose-inhibition (glucose-mediated suppression of genes under control of lac-UV5-promoter) can be used to suppress expression in pET-systems. I have tried to...
24 May 2014 3,394 3 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 4,921 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 9,627 4 View
I have two freely diffusing chromophores in aqueous solution and need to proof that these two are actually a FRET-pair. To achieve this, I suppose I would differentiate FRET from static and...
30 January 2014 6,126 23 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 1,170 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 2,247 42 View
