- → K.B.'s Content
There have been 19 items by K.B. (Search limited from 06-August 19)
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.
Whoa... You work in the lab and you seriously ask this kind of questions?... *facepalm*
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?
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.
Correct. (However, be careful with using high pH with proteins, they may not be stable.)
* 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?
Yes, 1 pH unit away from pI could be OK for binding to ion exchanger (go 2 units if you want to be sure).
OR if use a cation exchanger, the buffer pH=8 is enough?
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.
*** 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???
Because it is neutral salt (doesn't change pH of buffer).
* It is known that increase in salt gradient releases the bounded proteins from ion exchange resins. why NaCl is used?
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.
Should it be dialysed? what should the dialysis buffer content be?
Tris HCl - buffering agent
** What is the aim of these chemicals used in these steps?
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
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.
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).
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.
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.
BioMiha - why macros? Format one graph and copy it, then just change the source data for the copies, formatting would stay the same.
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.
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.