Phenol/Ether Protein ppt : Phase Inversion?

mlysik at hsc.vcu.edu mlysik at hsc.vcu.edu
Mon Dec 4 16:39:34 EST 2000


Hi all, maybe someone can help me out a bit.  I've been trying to concentrate some dilute protein solutions for SDS-PAGE and have been testing different methods which I've seen referenced in this newsgroup.  So far, I've tried the MeOH/Chloroform extraction, which seems to work ok except that I need to use 1.5mL tubes, which makes the process too time consuming (starting with 11 1-mL samples). 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



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