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K.B.'s Content

There have been 19 items by K.B. (Search limited from 20-February 19)

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#132854 70% ethanol in tap water...

Posted by K.B. on 14 April 2012 - 11:00 AM in General Lab Techniques

It may be equally economical for you to use medical-grade sterile water eg. water for injection or water for irrigation.

You don't need to use absolute ethanol. I was perfectly fine using low grade denatured ethanol (one denatured with methanol, acetone, or isopropanol - which actually makes it stronger disinfectant).

Also you could comparing cost to commercially available disinfectants like "Klericide" etc.

#132829 70% ethanol in tap water...

Posted by K.B. on 14 April 2012 - 03:13 AM in General Lab Techniques

Why should you boil the water?
Tap water should be pretty clean, so I imagine you wan to boil it to remove other componets?

And why should you, in the first place, use dd, DI or milliQ water? Whats wrong with tapwater?

Whoa... You work in the lab and you seriously ask this kind of questions?... *facepalm*

#132826 70% ethanol in tap water...

Posted by K.B. on 13 April 2012 - 11:56 PM in General Lab Techniques

Of course you can. Real question is - should you? :) Your lab must have distilled, dd, DI or MilliQ water source - better use one of those. If for some mysterious reason you can't do this - autoclave your water first. If you can't autoclave - at least boil it.

#122922 a few questions about ion exchange chromatography

Posted by K.B. on 02 November 2011 - 01:02 PM in Protein Expression and Purification

The isoelectric point of the desired protein is 8.91. I know if the buffer pH is above protein's isoelectric point, protein is charged negative. If it is below the isoelectric point, protein will be positively charged.


* If I use a buffer with pH=10 and a weak anion exchanger (DEAE-I have only anion exchanger) and then, decreasing the buffer pH gradually to slightly below the isoelectric point, can i take the desired protein?

Correct. (However, be careful with using high pH with proteins, they may not be stable.)

OR if use a cation exchanger, the buffer pH=8 is enough?

Yes, 1 pH unit away from pI could be OK for binding to ion exchanger (go 2 units if you want to be sure).

*** What should the content of the buffer be? for example, 5mM Na2HPO4, 1mM EDTA, 100mM NaCl and to obtain a pH=10, i will add NaOH to buffer solution???

This solution would not have any significantbuffering capacity at pH 10. I would rather try sodium carbonate buffer. However, if you want to use pH gradient elution it may not work properly with carbonate buffer. Gradient elution is quite tricky to perform properly, it requires buffer with wide working range, usually a mix of several (3-7) components instead of usual two. That's why most people use simple salt gradient.

* It is known that increase in salt gradient releases the bounded proteins from ion exchange resins. why NaCl is used?

Because it is neutral salt (doesn't change pH of buffer).

Should it be dialysed? what should the dialysis buffer content be?

It is not absolutely required but you may want to do this. It would be best to dialyse to buffer in which your protein would be most stable. Phosphate buffered saline (PBS) is quite good.

** What is the aim of these chemicals used in these steps?

Tris HCl - buffering agent
NaCl - neutral salt, proper ionic strength of solution
Urea - chaotrope, denatures protein, destroys higher order structure of proteins
Guanidine hydrochloride - chaotrope, denatures protein, destroys higher order structure of proteins
Triton - detergent, solubilizes proteins
DTT - reducing agent, destroys disulphide bonds
Na2HPO4 - buffering salt
EDTA - chelating agent, binds metal ions
Glutathione - antioxidant, protects proteins from radicals

#122857 ion exchange chromatography

Posted by K.B. on 02 November 2011 - 12:05 AM in General Lab Techniques

Your pH is OK.

Some proteins require salt or they will precipitate.

Amount of acceptable salt in sample (and mobile phase A) would depend on at what salt concentration it actually elutes during IEC. I.e. if your protein elutes early, you should keep salt concentration as low as possible; if it elutes late you may increase salt concentration in sample and mobile phase A.

#121825 problem with loading the protein on to SDS-Gel

Posted by K.B. on 18 October 2011 - 11:07 AM in SDS-PAGE and Western Blotting

1. Can't you use sodium chloride or lithium chloride for elution?
2. You could try buffer exchange with dialysis, ultrafiltration or gel filtration (eg. PD-10 columns).

#121310 HPLC mobile phase preperation?

Posted by K.B. on 11 October 2011 - 09:15 AM in General Lab Techniques

You have given ratios so final volume is not important.

Normally I would to this like that: 250 mL of methanol measured with 250mL cylinder, 750 mL of water measured with 1000mL cylinder (or 500mL + 250mL if you want to be bit more precise), 3 mL of acetic acid measured with 1000 uL pipette (3x).

If extra precision is required I would use volumetric flasks - 250mL of methanol in 250mL flask, transfer to 1L flask, fill up with water, add 3 mL of acetic acid (3x 1000 uL pipette).

#120344 how to convert pixel to micrometer ?

Posted by K.B. on 25 September 2011 - 12:52 PM in General Lab Techniques

40x magnification only means 40 units under the microscope equals to 1 unit in macro scale - it doesn't transform into pixel-micron relationship. (And by the way - to get real magnification you need to multiply by magnification of ocular lens and any other optical element between objective and camera that has magnification written on the body.)

#120340 how to convert pixel to micrometer ?

Posted by K.B. on 25 September 2011 - 11:02 AM in General Lab Techniques

If he knows the microscope settings he used for making those pictures, he could try taking a picture of an object with known dimensions and use that to calculate the scale.

#119476 Protein purification buffer

Posted by K.B. on 12 September 2011 - 08:49 AM in Protein Expression and Purification

I can do this for you - what pH and concentration you require? Do you need specific ionic strength? What would be the temperature you're planning to use this buffer at?

#118327 Is it possible to raise an antibody against a 11aa peptide?

Posted by K.B. on 26 August 2011 - 10:27 PM in Immunology

I've seen people raising antibodies against 5-7 AA peptides by conjugating them with carrier protein.

#115300 Free software for pKa calculation from chemical structure?

Posted by K.B. on 17 July 2011 - 06:42 AM in Be a Geek


I'm no longer in academia so I can't count on getting cheap licence and my company won't pay for the licence so I can check out my hunch. :)

Those are kinda new compounds - products of pre-column derivatization (for HPLC) of rather rare amino acid derivative and several interfering compounds. Knowing their pKa I could attempt to optimize resolution without resorting to trial and error or guessing.

#115294 Free software for pKa calculation from chemical structure?

Posted by K.B. on 17 July 2011 - 03:53 AM in Be a Geek

Howdy folks! :)

I am looking for software (preferably free) to calculate pKa for several compounds based on their chemical structure (from file or drawing by hand).

Chemicalize.org seems to have that option but it still in the beta stage and all I'm getting is errors and crashing browser when I try to enter new structure.

Help? :)

#115293 Alternative to Excel for charts

Posted by K.B. on 17 July 2011 - 03:49 AM in Be a Geek

I'm another person to recommend GraphPad Prism - it has some nice tools for data manipulation, easy statistical analysis, makes neat graphs and what is useful - it allows you to export them to format like TIFF required by most of journals.

BioMiha - why macros? Format one graph and copy it, then just change the source data for the copies, formatting would stay the same.

#115067 Protein stability at room temperature

Posted by K.B. on 14 July 2011 - 10:47 AM in Protein Expression and Purification

I don't know temperature stability of a protein I was working on for last couple of years and you expect me to know yours? :)
Check the literature data.

#115062 Protein stability at room temperature

Posted by K.B. on 14 July 2011 - 09:49 AM in Protein Expression and Purification

The answer is - it depends on the protein.

#114886 ask for advice on peptide mapping

Posted by K.B. on 12 July 2011 - 10:15 AM in Protein and Proteomics

I think you have precipitation because your reduced and alkylated antibody is randomly refolding in buffer without urea or guanidine hydrochloride. Check how much urea or Gu-HCl your trypsin can withstand and exchange into buffer which contains one of those in amount within acceptable limit.

Do you have amino acid sequence of your antibody? If yes - make a theoretical digest (eg. using Expasy PeptideCutter) - that would give you the "correct" number of peaks. By checking AA composition you can tell how many of those would be visible on 280nm (while you see all of them on 210-200nm). I would assign those peaks starting from the largest (if you have only UV data) and say everything else is just a product of random and unspecific digestion.

You should distinguish between sequence change and sequence coverage (ie. how much of the sequence is actually detected) by the method. I mean - depending on the digestion products and chromatography condition you may loose some minor peptides which would lead to less than 100% sequence coverage - it does not however indicate actual change in the sequence.

Personally I would not state the change in the sequence without MS (or better - MS/MS), unless there's major change in HPLC-UV profile.

#108038 Understanding graphs: where to start?

Posted by K.B. on 26 April 2011 - 02:04 PM in Bioinformatics and Biostatistics

Do you know that book well? Does it contain much easy to understand and realistic examples?

I need a good, easy to understand, book on statistics but dont really know any good titles.

No, but it is one of the very few with promising title.

Thank you for your reply and suggestions.
I took a look at the book suggested, however it does not include any figure which explains how to create and understand scientific graphs.
I also agree on the need to take a statistics course, but for the time being and as I am currently reading few papers urgently, I need some references or reading explaining this particular point "How to interpret the different symbols used in scientific figures?" Of coure, every other suggestions for later, when I have more time, to strengthen my statistics background is thankful.

The problem is that without any background in statistics basic explanation won't actually explain anything to you.

#107884 Understanding graphs: where to start?

Posted by K.B. on 25 April 2011 - 03:34 AM in Bioinformatics and Biostatistics

It seem you haven't got your statistics course yet. I would advise you to get one ASAP or at least read some books eg. this one.

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