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Questions related from Tomáš Hluska
When I get results from sequencing, they often overlap only partly or not at all. In such case it's easier to align them with the "parental" sequence (of what I expect), to see where they align....
02 February 2020 8,467 3 View
Hello everybody, I wish to separate apoplast from the rest of the plant (i.e. intact cells) to see whether the compound I treat the plants with goes inside or stays outside. When I mentioned this...
08 August 2019 3,003 3 View
How do I make a dot plot like in this paper Show the data, don't conceal them in Figure 1? All I'm finding on the Internet are only this type of dot plots:...
06 June 2019 2,283 5 View
I express my protein in pTYB12 vector as fusion with intein and then try to purify on chitin beads. Typically, I load overnight at low flowrates (something like 0.3 ml/min). The loading buffer is...
05 May 2019 2,716 7 View
I'm working with plant hormones cytokinins - derivatives of adenine with a side chain at the N6-group. They are mostly active as free bases (with no modification on the adenine core), or maximally...
03 March 2019 3,430 11 View
My first question concerns lupinic acid - conjugate of adenine derivative with alanine https://commons.wikimedia.org/wiki/File:Lupinic_acid.svg The question: What type of bond is it between the...
09 September 2018 9,048 0 View
Hi everybody, I'm currently expressing proteins in and extracting from bacteria. The procedure is that I pellet the bacteria from bioreactor, resuspend in buffer (50 mM Tris/HCl, pH 8.0; 1M NaCl;...
08 August 2018 2,562 9 View
Hello everybody, I'm currently growing E. coli in bioreactor and I have experience only with P. pastoris in bioreactor, so I'm not sure, how should it behave. With Pichia, one can try hard to...
03 March 2018 9,489 4 View
Some old papers (from the 70s) claim, that the substitution at position 7 is rare in the nature https://www.ncbi.nlm.nih.gov/pubmed/486500https://www.ncbi.nlm.nih.gov/pubmed/4640371 However, that...
10 October 2017 7,941 2 View
In the texts, it's often written, that the ligand has affinity towards a protein (with appropriate affinity constant). However, to me it always occurred that the protein binds the ligand and...
10 October 2017 7,166 0 View
Some time ago (my guess would be +-2 years), there was a paper where they showed that some bacteria are able to transfer from maternal plant to seed and to the new plant. I think I saw it only at...
09 September 2017 6,141 0 View
As you probably know, most position offers require at least three reference contacts. I believe I saw somewhere also, that they should not be older than three or five(?) years. And I suppose they...
05 May 2016 7,040 0 View
For example here http://www1.lsbu.ac.uk/water/enztech/nomenc.html they have D-xylose ketol-isomerase g-L-glutamyl-L-cysteine:glycine ligase b-D-glucose:oxygen 1-oxidoreductase L-histidine...
01 January 2016 5,030 0 View
Hello everybody, recently, I have problems with durability of my HPLC columns. Earlier, it lasted at least 3 months, sometimes up to six (that good at our department, often the columns used by...
12 December 2015 332 26 View
I want to measure enzymatic reaction with nucleoside phosphates, stop the reaction with methanol and then measure the NTPs on C18. For the separation I need 1% or lower methanol concentration (I'm...
11 November 2015 2,840 7 View
We use the Agilent's ZORBAX Eclipse Plus C18 RRHD 2.1×50 mm 1.8u columns, now for several years. I use K-phosphate buffer as A and methanol as B. This particular column was taken new recently,...
10 October 2015 2,825 4 View
Imagine the following - having lines of Arabidopsis overexpressing certain protein I want to measure the activity. From one line I will collect leaves from several plants separately (biological...
10 October 2015 8,726 3 View
I use HPLC to measure FAD to FMN conversion by my enzyme. The conditions are as follows: A: 15 mM formic acid, pH 4.5 by NH3 B: MeOH column ZORBAX Eclipse Plus C18 RRHD 2.1x50mm 1.8 micron in the...
09 September 2015 7,881 6 View
I want to measure, whether there is one compound in the extra-cellular matrix. I know there is plenty of it inside cells. How shall I proceed to be sure I do not break the cells? If I crush the...
09 September 2015 5,818 2 View
First, I'll describe what I'm doing, so you have more information than you need:) I measure FAD hydrolysis by HPLC. I mix McIlvaine buffer (phosphate+citrate), FAD, magnesium, DTT and my enzyme....
08 August 2015 3,304 15 View
Hi everybody, I'm talking about these http://tinyurl.com/Amicon-filters As I use them almost every day, I must re-use them. But since I don't want to lose my sample (obviously:), I always check...
08 August 2015 4,724 10 View
Let's say I have one plasmid (pPICZalpha) with one GOI linearized by one RE and I get 20 colonies. Does it make sense to screen them all for expression or is it basically nonsense? The point is,...
03 March 2015 8,403 6 View
I'm getting some unexpceted readings for my pO2 measurements. Could it be, that it reacts with something else than oxygen? I suppose not with peroxide, but what about methanol?
03 March 2015 8,466 10 View
Here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1375232/#__sec3title they write "The ‘e’ was dropped when it [thiamine] was found that it was not an amine." How is it so that thiamine is not an...
11 November 2014 5,995 6 View
So the enzyme activity is calculated as either increase of product concentration [P] or decrease in substrate concentration [S]. In practise, it's determined often using e.g. spectrophotometer as...
11 November 2014 5,496 3 View
So I have an enzyme with pI 5.5. I'll try to produce it in Pichia in medium with pH 4.0 or 4.5 as it grows better. However, as I perform purification in basic pH (7-8), I want to change the pH of...
10 October 2014 1,344 11 View
My advisor told me he had some sheet with ad for pipette tips used to cut bands from gel. However he didn't have it anymore so he couldn't tell me the official name nor brand. Have you ever seen...
09 September 2014 3,370 7 View
Which buffer do you use and why?
09 September 2014 2,891 13 View
When I grow Pichia and express my protein extracellularly, I concentrate the media in spin-columns and there's always some black mucus or slime or whatever. What is that? And is there any way how...
09 September 2014 9,233 3 View
I've cloned my GOI into several vectors creating fusion proteins with several tags: MBP, intein, His on N-terminus, His on both termini. Although I have activity in my extract, I'm not able to...
06 June 2014 972 9 View
We're running genotyping PCR with gDNA from rice and observed the results in the attached picture. There are two different setups: 1) for one locus we're running samples from 1 WT plant (marked...
04 April 2014 6,034 10 View
Since Triton is a non-ionic detergent, it should not bind to ionexes such as ResourceQ, right? So in principle it should be possible to use ResourceQ chromatography to get rid of excess Triton, right?
04 April 2014 1,431 5 View
To remove STOP codon I've cut my plasmid, blunt-ended (actually I'm going to do that) and ligated to itself. The question is, in case that two molecules of plasmid would join together (in the...
03 March 2014 1,937 12 View
I've recently run chromatography on Strep-Tactin column, but the activity was only in flow through fractions (75% total; no activity in elution). The sample was in 200 mM Tris/HCl pH 8.0 with...
02 February 2014 4,609 12 View
So I found the publication attached, where they describe protocol to transfer proteins of wide range in the means of two-step transfer. First they use 1.2 mA/cm2 for 1 hour to blot the small...
02 February 2014 9,351 0 View
I'm currently fighting with the Strep-tag/Strep-tactin chromatography. I observed, that the HABA is not eluted completely even by 100 mM Tris base (pH ~10.5). After the loading of proteins, I...
02 February 2014 8,245 14 View
There are different protocols for electrophoresis and blot considering the running conditions and it seems that everybody is doing it differently. Even the commercial kits recommend different...
12 December 2013 4,017 4 View
Do you know some enzyme that consequently isomerases and oxidases a substrate? what class would it be? Oxidase?
12 December 2013 3,086 3 View
On another question I was told, twice, I should not prepare my phosphate buffer by titrating dihydrogen phosphate to pH 7 or 8. I don't see any problem, because the phosphate will dissociate in...
11 November 2013 2,589 5 View
What if you have Triton X in your buffer and you need to concentrate your protein sample? Because Triton has micelles of size about 90 kDa, one cannot use gel filtration chromatography, or...
11 November 2013 9,345 8 View
I usually don't but since I'm about to use zeocin, I thought I'll make it "by the rules". However, because I do not remember what value it should be titrated to, I checked the internet and found...
06 June 2013 5,046 8 View
I noticed it already some time ago, that in accordance to suppliers MSDS some enzymes have actually higher activity in other-than-supplied buffer. Why do they not provide the best buffer? If you...
05 May 2013 4,038 4 View
Yesterday, I salted out my proteins (basically crude extract from maize) and dissolved in 1/10th volume buffer. I wanted to desalt it on HiTrap column so I filtered it through 0.22 um filter. When...
05 May 2013 4,559 15 View
I performed purification on FPLC hydroxyapatite column (CHT5-I from Bio-Rad) last week. I attached the chromatogram. Running conditions were as follows: buffer A: 5 mM KH2PO4; pH 8.0; 0.15 M NaCl;...
05 May 2013 2,346 7 View
I wanted to perform IMAC with Cu-imidodiacetic acid using 50 mM Tris, 0.15M NaCl and 15 mM bME, but the color vanished. I'm not sure what color Cu(I) is (now I found that CuCl is white, so it...
05 May 2013 5,167 8 View
I recently concentrated my partially purified protein (from maize flowers) about 13-50 times and the total activity increased about 2-3-fold. I was thinking about reasons of this and I could come...
05 May 2013 6,144 33 View
I got this text in a lab report of one student and I don't like the part about size of the plasmid, but I'm not sure if it's not correct, so if there is someone more experienced with this I'd be...
04 April 2013 3,895 1 View
I'm reading Guide to Protein Purification (From Methods in Enzymology series) and there is: "However, it must be pointed out that the concentration of the fluorophore cannot be calculated from...
04 April 2013 2,319 18 View
Which reducing agent at what concentration do you prefer in protein handling buffers for extraction, chromatography, storage etc. I know that largely depends on the protein in question, but I just...
04 April 2013 6,013 30 View
Based on what I found on the internet http://en.wikipedia.org/wiki/Murashige_and_Skoog_medium plantphys.info/plant_physiology/labdoc/tissueculture.doc MS and MSO media are the same thing? Or to...
04 April 2013 6,510 5 View
I'm studying protein crystallography now and I reached an inevitable point where I had to deal with the phase problem. Well, my problem is, that I'm not exactly sure what the phase is because no...
02 February 2013 8,230 6 View
I'm trying to stabilize my protein by use of different buffers. It obviously loves alkaline pH, because below pH 6 the activity was gone in few days even when stored at -80°C. However, even at pH...
12 December 2012 3,126 16 View
We are preparing Bradford reagent for our teaching at lab, but it's not working well currently. Do you have a working protocol?
10 October 2012 1,408 7 View
I have experienced that when I run on HPLC for several days non-stop (either non-stop loading samples or alternation of loading samples and low-flow stand-by) that the back-pressure increases. I...
10 October 2012 4,679 62 View
I've found on Wikipedia, that the critical micelle concentration of sodium dodecylsulphate is 0.0082M, what is in rough agreement with value from this...
08 August 2012 8,556 5 View
I'm trying to purify a novel enzyme, but I have problem, because it is unstable even at 4°C, so I have lost all the activity (there wasn't much even in the beginning) after only three...
06 June 2012 6,550 11 View
I have read that for SEC the volume of sample should not be more than 1-2%. But now I'm doing desalting on Sephadex G-25 (3x50cm column, dead volume is ~180 ml). I guess I can apply more 2%,...
06 June 2012 6,797 0 View
I have purified an enzyme and I need to identify it. People at our department are not able to do it always for some reason. Recently they've got some peaks, but were not able to identify the...
05 May 2012 8,135 12 View
I've always thought that SDS is supposed to denature proteins, destroy all non-covalent bonds etc. After reduction of disulfide bonds one should get respective polypeptide chains. However, after...
04 April 2012 8,315 39 View
I have been currently currently cloning one gene into pTYB12 vector and we had troubles with it all the time. Anyway, because we got no expression, I was currently trying to delete few first amino...
01 January 1970 7,681 5 View
Hi, a friend of mine found the following question in preparation tests for medicine: How much water do you have to add to 1 ml of solution with pH 8 to reach pH 5? I assume they want simply...
01 January 1970 3,213 5 View
