Lipid removal pre-SDS-PAGE

Peter Pediaditakis pxpst2 at vms.spam.suxs.cis.pitt.edu
Thu Feb 19 15:56:18 EST 1998


In article <6ci245$85m$1 at agate.berkeley.edu>, lhom at nature.berkeley.edu
(Louis Hom) wrote:

> What's the best way to remove fats from a protein sample before running it
> on SDS-PAGE?  I need to run some insect fat body samples and it seems like
> the fats are interfering with the run. 

Phenol Chloroform extraction.  Works well with extremely dilute or
concentrated samples.

Protocol:

Extraction of Dilute proteins from Solution

Step 1  Add an equal volume of phenol to protein containing sample ( up to
0.5 phenol).  For samples that are greater than 0.5 ml and less than 1.0
ml add 0.5 ml phenol.
Step 2  Vortex for 20 sec at the maximum speed
Step 3  Centrifuge for 5 min. @ 12000g  and DISCARD THE UPPER PHASE.
Step 4  Add 2 vol. of diethylether (no more than 1.0 ml) relative to the
phenol phase.
Step 5  Repeat step 2 and step 3.
Step 6  Repeat step 4 and step 5
Step 7  Dry the lower aqueous phase in the Speed Vac.  Samples can be
readily hydrated for further analysis.



reference:  Concentration of Dilute Protein for Gel Electrophoresis, Anal.
Biochem., 226, 382-383 (1995)

Hope this helps 
Peter Pediaditakis

-- 
"Don't you eat that yellow snow
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