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Concentrating protein lysates

Concentrating protein lysates - Protocols and Methods Forum

Concentrating protein lysates - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-02-2007, 11:27 AM
Angela Alexander
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Default Concentrating protein lysates



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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  #2  
Old 07-02-2007, 08:28 PM
AK
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Default Concentrating protein lysates

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" <[Only registered users see links. ]> wrote in message
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  #3  
Old 07-03-2007, 12:04 AM
DK
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Default Concentrating protein lysates

In article <[Only registered users see links. ].net> , Angela Alexander <[Only registered users see links. ]> wrote:

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

DK
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  #4  
Old 07-10-2007, 07:53 PM
Dr Engelbert Buxbaum
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Default Concentrating protein lysates

Am 02.07.2007, 20:04 Uhr, schrieb DK <[Only registered users see links. ]>:



You probably mean the Wessel & Flügge method, which uses methanol and
chloroform. Alternatively, one might use phenol/ether (Sauvé et al.)
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  #5  
Old 07-10-2007, 08:48 PM
DK
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Default Concentrating protein lysates

In article <[Only registered users see links. ].rossu.loc>, "Dr Engelbert Buxbaum" <[Only registered users see links. ]> wrote:

Yes, of course. Where did that "m" go?


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