Hi Stan,
When I used BSA in my TCA precipitation, I did not try to load the
whole sample on a gel afterward. But I agree with you that 1 mg would
probably be too much to load in one well (in my experience, the limit
is usually ~100 ug).
Could you immunoprecipitate your protein?
Alternatively, you could TCA-precipitate the proteins, bring them up
in a smaller volume, and then filter the sample through a 10,000 or
30,000 MWCO centrifugal filter. The flow-through would contain only
small proteins, including yours, but BSA would be excluded. Ideally,
you could avoid the precipitation altogether and just filter the
sample, but this will not concentrate the flow-through and it sounds
like you really need to concentrate it too. So the two-step procedure
would be my best suggestion. I have never seen the big lump that you
describe so I can't help with that part.
Hope that helps,
Irit
On Mar 14, 2010, at 8:33 PM, Stanley Cheung wrote:
Hi Irit and everyone,
I heard BSA helps if the sample has very low concentration. I just
wonder if the high BSA concentration would affect the later SDS-
PAGE. Let say if you need to add BSA to 1mg/mL in 1mL sample.
Finally, need to add all the sample for running SDS-PAGE in one
well. So there is at least 1 mg of total proteins (Or 1 mg BSA plus
negligible amount of proteins from the original unconcentrated
sample).
There are two problems I may concern. First, whether the lump
formed by high concentration of BSA can be dissolved in sample
buffer. Second, whether sample with such a high protein
concentration would affect the resolution of the SDS-PAGE, esp for
protein with small molecular weight. I tried to load 250micro gram
total protein extracted from brain lysate and resolve it by SDS-
PAGE, however, the resolution is very bad, esp at small molecular
weight. My target protein is 4kD and I used 16.5% Tricine gel. The
reference for this experiment was mentioned in Lesne S. et al.,
2006, Nature, v440, 352-357 and its supplementary methods. But I
never get it works.
Best,
Stan
________________________________
From: Irit Rappley <irapp...@scripps.edu>
To: Nikola Wenta <nikola.we...@nottingham.ac.uk>
Cc: "meth...@oat.bio.indiana.edu" <meth...@oat.bio.indiana.edu>
Sent: Fri, March 12, 2010 2:53:50 AM
Subject: Re: Protein precipitation - acetone?
Some people add 1 mg/mL BSA to their sample in order to get the
total protein concentration high enough for precipitation. Of
course, then you're left with lots of BSA in your precipitate too....
On Mar 11, 2010, at 9:27 AM, Nikola Wenta wrote:
Hi Iraz!
I don't have any clue about acetone precipitation of proteins, but
generally, precipitation with saturated Ammonium sulfate solution
provides a good means to concentrate proteins and to get them into
a save state for short-term storage. Unfortunatelly, you would
already need the protein solution to be at > 1 mg/ml in order to
get it to precipitate. Thus, in your case this method doesn't seem
suitable. You could also try to concentrate the protein with
Centricons, but you would rather loose protein to the membrane
than concentrate your solution as it is already too diluted. Why
not loading maximum volume into biggest possible pockets on a gel
with maximum thick spacers? Additionally you could use a
acrylamide percentage that "compresses" your protein band, giving
you a better signals in WB.
Best, Niko
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requ...@oat.bio.indiana.edu
Gesendet: Do 11.03.2010 17:03
An: meth...@magpie.bio.indiana.edu
Betreff: Methods Digest, Vol 58, Issue 7
Message: 8
Date: Thu, 11 Mar 2010 16:56:05 +0100
From: "Iraz Toprak Aydin" <iraz.ay...@epfl.ch>
Subject: Protein precipitation - acetone?
To: <meth...@magpie.bio.indiana.edu>
Message-ID: <004401cac133$5b7c0df0$127429...@aydin@epfl.ch>
Content-Type: text/plain; charset="us-ascii"
Dear all,
I have to do a western blot, but my protein concentration is very
low, and I
have to run a mini gel. So I was thinking of precipitation the
proteins. I
have never done this before. Does acetone have a bad effect on the
blotting?
Are there any points that I should be careful about?
Thanks in advance...
Iraz Toprak Aydin
EPFL SV ISREC, Station 19
Batiment SV, SV 2540
CH-1015 Lausanne
Switzerland
Tel: +41 21 693 07 36
e-mail: <mailto:iraz.ay...@epfl.ch> iraz.ay...@epfl.ch
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