azo
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extracting PDC protien
I dont want to rehash old threads but i would like to understand more about alchohol dehydrogenase complex .
There is lots of information out there about different ways to extract pdc protien from c utilis which i have selected due to it being resistant to
toxic substrates.
And has anyone had experience doing this that could put light on the best way to proceed foward.
!!! what in the hell is the differance between whole cell pdc and purified pdc.
any help would be very much appreciated as i no very little about protien chemistry
regards azo.
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Sauron
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Where's chemoleo? He's the resident biochemist.
You are talking about extracting an enzyme.
Sounds like a job for HPLC to me.
Preperative, nonmetallic HPLC at that like a Waters 650 Advanced Protein Purification System. PTFE pump heads so no metal ion contamination of the
enzyme. The injectors are nonmetallic too.
BTW it's protein not protien.
Sic gorgeamus a los subjectatus nunc.
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Maya
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ahem, if I may.
you need to completely homogenize , your liver sample I presume, with a blender or dounce homogenizer.
then, filter the stuff thru cheesecloth. centrifuge the supernatant at high speed and keep the supernatant discarding the pellet assuming your enzyme
is in the cytoplasm. ppt with sat. ammonium sulfate. all at ice cold. then you need to do some
ion exchange chromatography on a big column. either anion exchange or cation exchange and elute your compound of interest ( I won't tell you which ,
anion or cation cause I ain't gonna look it up ). then check your fractions for which ones exhibit ADH activity by assay enzymatically ( a
spectrophotometer is handy ). thats exactly how you do it . but I don't have the exact protocol. straight from 1st year biochem
forgot to add the centrifugation step, very important!
[Edited on 24-4-2008 by Maya]
\"Prefiero ser yo extranjero en otras patrias, a serlo en la mia\"
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roamingnome
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Having just obtained an International 6000 RPM centrifuge i am again excited about purified PDC protein
Not sure if wheat germ PDC is the most hardy as compared to c utilis, but the ease at getting it seems unparalleled.
http://www.jbc.org/cgi/reprint/196/1/375.pdf
Improved purification of pyruvate decarboxylase from wheat germ...
http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1432-1033...
See you must lysis the yeast cells but with wheat germ you could have crude protein in a few hours.
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azo
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Thanks for the info maya i take it you mean ppt with ammonium sulfate untill the enzyme just starts to ppt and then centrifuge correct me if i am
wrong. I suppose i need to no the net charge on the pdc enzyme whether it is anion or cation any help with this info would help a lot.
roamingnome like you said i dont no if pdc from wheatgerm is as hardy as c utilis but for what i am doing from all refs it seems that pdc from c
utilis seems to be the best and they also state that whole cell pdc performs much better than purified pdc. Proberly due to the protection from toxic
substrates from other cells which are not there on purified pdc correct me if i am wrong .
Thanks for the tip on wheatgerm i will do some research on it.
! And thanks for the replies
regards azo
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chemoleo
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Ok, first off HPLC is definitely not the method of choice, because HPLC works under conditions (i.e. acetonitrile,IPA or methanol with 0.1 % TFA)
where proteins unfold, meaning that activity is lost due to loss of the native fold. Essentially you end up with a polypeptide of random
structure rather than the defined original structure of the native folded enzyme.
So you have to use buffers - usually isotonic of some kind (50 mM K phosphate, 100 mM NaCl pH 7.5 springs to mind).
Most proteins are happy under these conditions.
Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of many purification
protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS concentration range, which is
the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation) are taken and the fraction that
contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school biochem.
Whole cell vs purified - naturally the whole cell extract is better because it contains cofactors and other proteins that you may not have present in
your purified extract.
Despite what biologists claim about knowing about a particular subject/reaction, much more is UNknown. A fact I found out the hard way. And there are
many crap biologists/biochemists, and I freely admit they wouldn't survive in other sciences.
Anyway, I can check up ADH (alcohol dehydrogenase) and PDC (pyruvate decarboxylase) in i.e. Methods in enzymology, but in the meantime a patent
search might useful.
If you give me the biochemical properties of ADH/PDC (MW, pI, solubility, etc), I can work out a purification scheme for you...
[Edited on 14-11-2009 by chemoleo]
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Maya
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<<<< Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of
many purification protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS
concentration range, which is the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation)
are taken and the fraction that contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school
biochem.>>>>>>>>>>
you know as well as I do that this is of course completely old school biochem. generalizing for all enzymes. No optimization given, as I said. find
out empirically the correct AS conc. and cationic or anionic properties. I forgot to mention centrifuge before and after AS ppt.
What other way does a lay person have of success?
The way it is done nowadays usually destroys activity but is much simpler, just lyse the sample, run it on an sds gel, transfer the gel to a membrane
and then western blot it. Or alternatively, after running it on a gel , then stain it, excise the band at the correct mol. wt. and run it on the
LC/MS.
But thats 2nd year biochem and pretty much too far above the laypersons means
But then the guy said he wants the entire complex that it binds to it as well? So then the best way is to immunoprecipitate it with your antibody and
sephadex A/G or equivalent
\"Prefiero ser yo extranjero en otras patrias, a serlo en la mia\"
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azo
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Well i have been called a lot of things before but never a lay person been layed a few times i hope this is what you ment .
Getting back to you chemoleo i will post the relevant info for you as soon as possible ! btw it don't seem that you are to optimistic about the said
subject/reaction.
thank you very much for all the help.
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MagicJigPipe
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Definition of layman from Dictionary.com:
2. a person who is not a member of a given profession, as law or medicine.
As you can see, it's not an insult. It just basically means you do not have advanced knowledge in a certain subject.
"There must be no barriers to freedom of inquiry ... There is no place for dogma in science. The scientist is free, and must be free to ask any
question, to doubt any assertion, to seek for any evidence, to correct any errors. ... We know that the only way to avoid error is to detect it and
that the only way to detect it is to be free to inquire. And we know that as long as men are free to ask what they must, free to say what they think,
free to think what they will, freedom can never be lost, and science can never regress." -J. Robert Oppenheimer
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zimaman
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Quote: Originally posted by chemoleo  | Ok, first off HPLC is definitely not the method of choice, because HPLC works under conditions (i.e. acetonitrile, DCM or methanol with 0.1 % TFA)
where proteins unfold, meaning that activity is lost due to loss of the native fold. Essentially you end up with a polypeptide of random
structure rather than the defined original structure of the native folded enzyme.
So you have to use buffers - usually isotonic of some kind (50 mM K phosphate, 100 mM NaCl pH 7.5 springs to mind).
Most proteins are happy under these conditions.
Maya, the procedure using ammonium sulfate precipitation, is this out of a text book referring to ADH or PDC specifically or one of many purification
protocols used for protein purifications in general? With ammonium sulfate, the target precipitates only at a certain AS concentration range, which is
the basis for purification! So usually steps of 0-0.5, 0.5-1.0, 1.0-1.5 etc M (NH4)2SO4 (or finer, upon optimisation) are taken and the fraction that
contains the protein (assayed by mass, activity, whatever) is then further purified....but this is old school biochem.
Whole cell vs purified - naturally the whole cell extract is better because it contains cofactors and other proteins that you may not have present in
your purified extract.
Despite what biologists claim about knowing about a particular subject/reaction, much more is UNknown. A fact I found out the hard way. And there are
many crap biologists/biochemists, and I freely admit they wouldn't survive in other sciences.
Anyway, I can check up ADH (alcohol dehydrogenase) and PDC (pyruvate decarboxylase) in i.e. Methods in enzymology, but in the meantime a patent
search might useful.
If you give me the biochemical properties of ADH/PDC (MW, pI, solubility, etc), I can work out a purification scheme for you...
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I am interested to know if you have made that procedure yet. If not, here are some properties of the proteins:
ADH:
M.W.: 38kD
pI: 6.79
charge at pH7: -1.4
PDC:
M.W.: 61kD
pI: 6.18
charge at pH 7: -6.2
I am interested in separating the 2 proteins in bulk and keeping the PDC containing portion. I wouldn't need to purify past that point. I just need to
exclude ADH. The organism would be saccharomyces cerevisiae.
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chemoleo
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Re bulk separations from crude extracts, I'd first do ammonium sulfate precipitation (thereby getting rid of all the fats, sugars, glycans etc), and
see if the two precipitate at different concentrations. They probably will. Then dialyse into a low salt buffer, pH 8.
Then, anion exchange (Q columns) at pH 8, see again if you can separate if it didn't separate during the previous step. Then, as a final step, you can
do size exclusion to really polish the proteins...
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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