Concentrating protein lysates

[Angela Alexander]

I'm working with a new cell line that's giving me very dilute lysates (even with a vastly reduced volume of lysis buffer), such that I can barely load 15-20 micrograms of protein on a western gel. I'm doing a lot of signaling analysis and sometimes need more protein than that to get solid blots for some phospho-antibodies (we usually load 30-40). So I was wondering if you guys had any suggestions on the best method for concentrating my lysates without affecting my data ie losing robustness of phosphorylation.

Speedvac'ing might be reasonable, as we have one in my lab right behind my bench, but I'm concerned the length of time the lysate will not be kept cold that will be required might not be such a great idea. Is dialysis the only way to go, or is there a quicker, easier method?....since I will be generating many long time courses/dose responses with these cells once I figure out the best method/something that works.

Thanks
Angela Alexander
Graduate Student
University of Texas MD Anderson Cancer Center

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[AK]

Angela, you might want to try ultrafiltration. Amicon (now i think part of
millipore) should have many choices. basically, you start with lets say 2ml
of ur lysate and can concentrate down to 50ul in relatively short time. this
is centrifugation based so no problem with temp. these units will come in
mol.wt cut offs, which means u choose one which will not let your protein of
interest pass through the membrane.

Also, what I have done in past (it is cheating i know!) is to let the sample
compact in stacking gel, then load again. the second load will catch up to
the first with sharp bands. this require u make ur stacking get just a bit
longer.

hth, good luck
AK

"Angela Alexander" <udfl...@yahoo.com> wrote in message
news:mailman.165.118339...@net.bio.net...

[Ozan Aygun]

Hi,

I have ben using "Strataclean Beads" from Stratagene
for concentrating small volumes of protein extracts
for western analyses. It is pretty efficient and time
saving. I do not remember how expensive the resin was
but you need very little per sample. Just add 5ul of
resin up to 50-100ul of protein extract and vortex
well to mix. Then spin down the beads and aspirate the
supernatant (simply captures protein mixture on the
beads). Boil the beads with desired volume of
SDS-sample buffer (1X) for 5 min and spin again. Load
the supernatant carefully.

These beads are also pretty useful for desalting
protein extracts that are in high salt solutions
before loading on SDS-PAGE analysis thereby preventing
precipitation.

Hope this may be any help, good luck,

Ozan

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[Dr Engelbert Buxbaum]

Am 02.07.2007, 20:04 Uhr, schrieb DK <d...@no.email.thankstospam.net>:

>> I'm working with a new cell line that's giving me very dilute lysates
>> (even with a vastly reduced volume of lysis buffer),

> Google for ethanol/chloroform precipitation. Just what you need.

You probably mean the Wessel & Flügge method, which uses methanol and
chloroform. Alternatively, one might use phenol/ether (Sauvé et al.)

[Deitiker, Philip R]

Lyophilization of the lysates.

You probably want to clarify the solution first by spinning and ultrafiltration.
The caveats of lypohilization is that the protein needs to be freeze tolerant. Lyophilizers typically reduce the temperature well below freezing, inside the chamber is -50'C, the outside is below -20C typically.

Many proteins can be resolubilized in pure water after lyophilization. The salt will concentrate however many fold and this will cause your gel lanes to sway. Therefore before lyophilization it is best to reduce the salt concentration if at all possible. One very fast way to reduce the amount of salt is a G-15 desalting column. This of course will also separate out small peptides, amino acids and the like. It can be done however at 4'C.

Ultrafiltration on an amicon LWCO membrane is another way to get rid of the salt and concentrate the protein at the same time.

Philip

[Dr Engelbert Buxbaum]

In article <ba8823a8-e4fe-4749...@googlegroups.com>,
bimjhanab...@g.harvard.edu says...

>
> On Monday, July 2, 2007 at 7:27:59 AM UTC-4, Angela Alexander wrote:
> > I'm working with a new cell line that's giving me very dilute
> > lysates
> >

> > Speedvac'ing might be reasonable, as we have one in my lab right
> > behind my bench, but I'm concerned the length of time the lysate
> > will not be kept cold that will be required might not be such a
> > great idea. Is dialysis the only way to go,

You can use the SpeedVac as the sample is kept cold by the evaporation
of water. Just stop as soon as all water is evaporated.

However, you may find chloroform/methanol precipitation (Wessel & Flügge,
Anal. Biochem. 138 (1984) 141-3) or precipitation by TCA with DOC as
carrier (Bensadoun & Weinstein, Anal. Biochem. 70 (1976) 241?50) much
simpler.

For larger samples dialysis against solid PEG is very effective (just
cover the dialysis bag with PEG 20K), but make sure that you do not allow
the sample to dry out completely (it's usually not possible to
reconstitute if that happens).

And of course, there are always centrifugal concentrators and (again, for
larger volumes) Amicon ultrafiltration cells (now flogged by Millipore,
IIRC).