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How to prep mouse urine samples for SDS-PAGE and Coomassie staining?


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#1 esbena

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Posted 01 December 2017 - 02:21 AM

Hi.

 

I'm working with a kidney disease mouse model, and we want to analyse some urine samples by SDS-PAGE and Coomassie staining. However, I'm concerned that the high salt and urea could interfere with the SDS coating of the proteins and the actual running of the gel.

 

Is it desirable to centrifuge the samples before ?? 2000g 10 min?? 

We don't want to interfere to much with the samples (by PEG8000 precipitation a.o.) because we want to be able to use them for other analysis.

 

Thanks

 

esbena



#2 mdfenko

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Posted 01 December 2017 - 04:19 AM

the salts will affect the profile generated in the gel. neither the salts nor the urea will interfere with sds binding. urea, in fact, helps sharpen banding in sds-page.

 

centrifugation will remove insoluble contaminants but not urea (or uric acid) or salts. to do that, you will have to either precipitate the proteins and resuspend in sds sample buffer or dialyze the urea and salts away from the sample.

 

bear in mind that the protein concentration may be very low and you may have to concentrate the sample prior to sds-page. you can use a membrane concentrator (eg minicon) to diafilter and concentrate the samples.


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#3 bob1

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Posted 01 December 2017 - 07:02 AM

Another thing to consider is that the Major Urinary Proteins (MUPs) in male mice might obscure bands from other proteins. It is best to work with females for urinary things in mice.






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