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SDS precipitates when I add my sample to PAGE sample buffer

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  #1  
Old 06-08-2006, 07:03 AM
scott.coutts@med.monash.edu.au
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Default SDS precipitates when I add my sample to PAGE sample buffer



Hi,

Does anyone know what could be causing the SDS to precipitate out of my
SDS-PAGE sample buffer when I mix it with my sample?

Some background:

I have a Triton x-114 membrane prep and i've used Amberlite
XAD-1180/Biorad Biobeads to remove most, but not all, of the detergent.
The solution I have left over is concentrated about 10 fold in a
centrifugal filter device. The sample is prepared in PBS+5mM MgCl2 at
pH 7.2, but after the prep the pH is about pH 10 (as is shown by
indicator strips). The solution is clear, with no precipitated
material. As soon as i put this in my 2xSDS-PAGE sample buffer
(standard stuff, 2% SDS, B-ME, bromo blue) it causes the SDS to come
out of solution. I tested that it was the SDS by adding some of my
sample directly to a 2% SDS solution and the same thing happens.

There's no guanidine, and no K+ salts in my sample, by the way!

Cheers,

Scott.

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  #2  
Old 06-11-2006, 04:11 PM
Dr Engelbert Buxbaum
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Default SDS precipitates when I add my sample to PAGE sample buffer

[Only registered users see links. ] wrote:


Phosphate buffered saline (PBS) contains potssium. In addition, check
the solubility of SDS in the presence of bivalent metal ions like Mg,
there might be a problem too.
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  #3  
Old 06-11-2006, 05:00 PM
Han
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Default SDS precipitates when I add my sample to PAGE sample buffer

Dr Engelbert Buxbaum <[Only registered users see links. ]> wrote in news:e6hfc1
$q3p$00$[Only registered users see links. ]:

Also, SDS precipitates easily in the cold, doesn't it?
--
Best regards
Han
email address is invalid
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  #4  
Old 06-13-2006, 12:56 PM
scott.coutts@med.monash.edu.au
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Default SDS precipitates when I add my sample to PAGE sample buffer

Hi folks,

Thanks for the ideas, but I dont think that it's either potassium or
temperature related.

I had already thought about the PBS and checked (experimentally) that
the concentration of K+ is low enough that it is not causing the
precipitation that I have seen. Well, it will not precipitate the
equivalent concentration of SDS, at least.

Also, it's not precipitating due to the cold, because the precipitate
does not dissolve on heating, and on boiling it tends to increase. THe
precipitate becomes more compact and settles more easily, in fact.

Cheers,

Scott.

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  #5  
Old 06-13-2006, 02:41 PM
Dr Engelbert Buxbaum
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Default SDS precipitates when I add my sample to PAGE sample buffer

Han wrote:


That depends on concentration. But the Lithium salt is used for low
temperature electrophoresis, because of its higher solubility.
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  #6  
Old 06-14-2006, 04:12 AM
scott.coutts@med.monash.edu.au
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Default SDS precipitates when I add my sample to PAGE sample buffer


Dr Engelbert Buxbaum wrote:

Yeah, my samples are not encountering a temperature cold enough to
precipitate the SDS anyway.

Cheers,

Scott.

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  #7  
Old 06-18-2006, 11:25 PM
Bean Long
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Default SDS precipitates when I add my sample to PAGE sample buffer

Scott,

SDS can easily drop out at extremes of pH. You mentioned pH 10?? High
concentrations of SDS will often drop out during acid precipitation but
can easily be removed by redissolving in neutral buffer or water prior
to acetone dropping, for example. Also, have you checked your SDS
concentration? If you can see a ppt then it could be more than 2%!?

Best of luck

[Only registered users see links. ] wrote:


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Bean

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