Protein precipitation - acetone?

[Irit Rappley]

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 <irappley from scripps.edu>
> To: Nikola Wenta <Nikola.Wenta from nottingham.ac.uk>
> Cc: "methods from oat.bio.indiana.edu" <methods from 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
>>
>> -----Ursprüngliche Nachricht-----
>> Von: methods-bounces from oat.bio.indiana.edu im Auftrag von methods- 
>> request from oat.bio.indiana.edu
>> Gesendet: Do 11.03.2010 17:03
>> An: methods from 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.aydin from epfl.ch>
>> Subject: Protein precipitation - acetone?
>> To: <methods from magpie.bio.indiana.edu>
>> Message-ID: <004401cac133$5b7c0df0$127429d0$@aydin from 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.aydin from epfl.ch> iraz.aydin from epfl.ch
>>
>>
>>
>>
>>
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> Irit Rappley, PhD
> The Scripps Research Institute
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