Could you add a tag to your protein (eg. polyhistidine can be pulled down with nickel)?
I'll send you an interesting research review paper on Reversed micelle solvents as tools of enzyme purification:
TY - JOUR
T1 - Reversed micelle solvents as tools of enzyme purification and enzyme-catalyzed conversion
JO - Biotechnology Advances
VL - 26
IS - 6
SP - 516
EP - 532
PY - 2008/11//
Y2 - 2008/12//
T2 -
AU - Tonova, Konstantza
AU - Lazarova, Zdravka
SN - 0734-9750
DO - http://dx.doi.org/10.1016/j.biotechadv.2008.06.002
UR - http://www.sciencedirect.com/science/article/pii/S0734975008000669
KW - Reversed micelle solvent
KW - Reversed micelle extraction
You mean how is it possible to purify different proteins with limited number of chromatography matrices?
Dear Tomas,
It is great that you have reached to the depth of question. In my opinion, sequence of the column application may be one major solution of this problem. It is absolutely a brain storming problem, and further solutions with merit are welcome.
Well, the thing is that you have combination of possibilities, so e.g. if you get 5 fractions from first and second column, 15 fractions from other two columns, you'll have 5*5*15*15 possibilities where particular protein can be. Thus there is high chance of cleaning your protein. Afterwards, with affinity chromatography you do not get that many fractions, but high selectivity works similarly like if you had high number of fractions (i.e. you purify e.g. 1 in 100 proteins)
Dear Thomas,
I am glad to read your well approaching answer reflecting your in depth awareness and updating of 'Enzymological techniques'. Further exploration along this particular notion is expected in future as well.
couple your column chromatography with 2D gels. It is the fastest and most accurate way to do it. Couple size exclusion chromatography with ion exchange if you have no access to 2D gels.
The way you do thousands of work with ten fingers with yours two hands
Dear Subhadrata,
I can understand what you mean to focus on. Although your statement is not scientific, I can dissect it out and conclude your approach. Being a Professor of Biotechnology, I can wish you all the best in your endeavor, and give my statement as follows
There are infinite number of enzymes in biological systems, their biochemistry is also different reflecting that their mode of action as well as purification would certainly be different. Thus, in my opinion, if the sequence of limited types of columns: Gel Filtration (Molecular Sieving); Ion Exchange (Anion/cation); Affinity is altered and repeated concomitant with incorporation of Single and/or Two-D-Electrophoreasis may probably be helpful in successful purification of various enzyme-proteins of metabolic interest(s). Besides, coordination between percent recovery of enzyme and removal of junk protein is also advised to be considered step-wise. These efforts may lead towards success in this context. At last, I would like to emphasize onto the point that 'Enzyme purification is a tedious job and absolutely hit and trial adventure'.
Thank you for the remark on my reply. Possibly you have not taken the essence of the comment (although you have repeated the same in the following massage what I stated ). Do you think that there is no science in the performance of of each of our fingers performing thousands of work ? I have spent 45 years of my research on protein purification and reported purification of at least 15 glycosidases. Thank you for your cordial wish to do the best in the domain
Dear Dr. Subhadra Sengupta,
Thanks a lot for your re-remark. In fact, I had no intention of saying what was reflected out by my statement. I reformat my statement and view as, "There is visible simili in your practical based statement to the 'Enzyme purification'. What I could judge is that coordination of fingers (although limited in number) is quite similar to that concerning with limited number of columns versus 'Enzyme Purification'. Further, being younger to you in terms of age as well as experience (mine one is more than 15 years in various domains of Biochemistry/Biotechnology with major reference to protein purification and characterization)., I honor to your previous as well as this very statement, and hoping that you would certainly be an ideal model of inspiration to me to move on with pace in this specific domain of Biochemistry/Biotechnology.
With kind regards,
Sanjay
My all best wishes for your success in research career in the domain of biotechnolgy
Dear Dr. Subhabrata Sengupta,
With thanks and kind regards,
Sincerely,
Sanjay
May be you can add a step like precipitation with Ammonium sulfate at the beginning.
Depends on your protein´s properties, but can be usefull.
precipitation with ammonium sulphate is usually not much selective. It's good to concentrate your sample in the beginning.
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