Fw: Phenol/Ether Protein ppt : Phase Inversion?

Peter Pediaditakis pxpst2 at dlt.pitt.edu
Mon Dec 4 19:46:09 EST 2000


In article <200012042247.eB4Ml8b18697 at ns.qnis.net>, dbell at qnis.net
("Deanne Bell") wrote:

> Hi Melanie
> I don't want to show my ignorance, but aren't chloroform and phenol used to
> purify proteins out of nucleic acid extractions? Woudn't they cause the
> proteins to precipitate/denature?
> Deanne Bell

Remember that presipitaion and extraction are very different. 
Preciptation occurs when the solution can no longer hold the solute and it
falls out of solution.  Extraction occurs when the solubility in one
solution is >>>>>>> than the other solution.  Straight Chloroform will
surely precitate proteins but Phenol is excellent at solvating or
extracting proteins.  
> ----------
> 
>> The phenol/ether extraction
>> looks to be moire ideal since the whole volume stays below 1.5 mL. 
>> However, upon attempting this method I found that the "upper layer" (which
>> is supposed to be my aqueous layer) is sometimes approx. 1-mL (the volume
>> of my starting sample) and other times approx. 0.5-mL (the volume of phenol
>> added.  This inversion occurs mostly with the higher salt fractions (0.5
>> and 1.0M KCl).  If I let the samples sit a minute on the counter top after
>> the centrifugation step, I start to get a white ppt throughout the lower
>> phase.  My questions are: (1) is this a phase invers!
>> ion and, if so (2) should I remove the lower layer, including the ppt,
>> and continue with the upper layer or should I remove the upper layer and
>> continue with the lower phase containing the ppt?
> > 
> > Thanks in advance for you help,
> > 
> > Melanie 

I have run into this problem as well.  The phases do not invert.  What you
are getting is salt precipitate it the interface.  Salt is not very
soluble in phenol.  If you are using unstaturated phenol then your phenol
pahse will increase in size due to the fact that some water will go to the
phenol phase.  Some salt will get carried into the phenol by the
water(regardless of whether sat. or unsat. phenol is used) and over time, 
the salt will precipitate from that phase into a white mess.  You must
remove the lower phase but as you have found, that is dilfficult because
there is no clean miniscus and the salt preciptate phase in between will
house some proteins.  Enough to mess with your yields significantly.


If I may ask, Why is there so much salt?  

Your only solution is to dilute your protein further and modify the method
to be a batch process(designe for 20 ml volume with 7-10ml of phenol. When
you get to the end following the two ether washes, you will have about 1-2
ml of protein in water.  repeat the extraction one more time and you will
have it in <50ul.

regards

-- 
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology

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