[Protein-analysis] Re: Glycerol during protein purification and

Lu Falong via proteins%40net.bio.net (by FLLu from genetics.ac.cn)
Sat Jul 21 00:38:46 EST 2007


proteins-request,

	   I have ever use glycerol at 25% concentration. It is fine. However, I have no idea if it could help with you problem.

======= 2007-07-21 01:08:54=======

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>Today's Topics:
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>   1. Glycerol during protein purification and protein
>      oligomerization state (Emily Arturo)
>   2. ghost" dots on blot (Ligong Wang)
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>----------------------------------------------------------------------
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>Message: 1
>Date: Thu, 19 Jul 2007 21:13:03 -0500
>From: "Emily Arturo" <ecgarturo from gmail.com>
>Subject: [Protein-analysis] Glycerol during protein purification and
>	protein	oligomerization state
>To: proteins from magpie.bio.indiana.edu
>Message-ID:
>	<28d2e0e90707191913m419bc7e7hb5deccfa261bdd02 from mail.gmail.com>
>Content-Type: text/plain; charset=ISO-8859-1; format=flowed
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>I am experiencing difficulty with reproducing the oligomerization
>state of a protein overexpressed and purified from E. coli, from prep
>to prep.  I have considered increasing the glycerol concentration in
>the buffers to promote the dimer state over the monomer state (no
>other oligomerization states are detected).  The DNA binding protein
>is likely not bound with DNA during these later stages of purification
>as we have incubated the cell extracts with a cocktail of nucleases.
>
>Does anyone have any suggestions?  The buffers already contain 10%
>glycerol; is there any reason (that I've not yet thought of) that I
>might not want to increase the glycerol concentration further?   I'm
>ashamed to admit it, but I'm currently uncomfortable with more than
>10% simply because I've not seen literature that cited a buffer
>containing more than 10%!
>
>Any other suggestions are welcome...
>
>Thank you,
>Emily.
>
>
>
>------------------------------
>
>Message: 2
>Date: Thu, 19 Jul 2007 12:42:33 -0700 (PDT)
>From: Ligong Wang <ligongw2004 from yahoo.com>
>Subject: [Protein-analysis] ghost" dots on blot
>To: proteins from magpie.bio.indiana.edu
>Message-ID: <332925.48082.qm from web52507.mail.re2.yahoo.com>
>Content-Type: text/plain; charset=iso-8859-1
>
>Did you block the membrane with BSA before you probe it with primary&secondary antibodies? If you did not, the primary Ab will bind to the membrane thus give higher background.
>
>ligong
>
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Sincerely,¡¡¡¡¡¡¡¡¡¡

Lu Falong
 Group 808
 Institute of Genetics and Developmental Biology, CAS 
 Beijing, PR China
 FLLu from genetics.ac.cn
 2007-07-21  




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