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Runing problems


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#16 Vemovied

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Posted 12 November 2013 - 01:24 AM

If you are simply doing an SDS gel, then you could dissolve in loading buffer (SDS or LDS). If that doesn't work, then dissolving in 4 to 8 M urea would surely work.

I would like to measure the protein with BCA before the SDS gel, so this Urea buffer, it is just urea? or a mix with other things (tiourea/sds/...)

Thanks thanks thanks so much!



#17 Vemovied

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Posted 12 November 2013 - 01:53 AM

If you are simply doing an SDS gel, then you could dissolve in loading buffer (SDS or LDS). If that doesn't work, then dissolving in 4 to 8 M urea would surely work.

I would like to measure the proteinf first to run the SDS gel, so I will need to have a suspendsion.

This urea buffer it is just urea or have somethng else?

Thanks Thanks Thanks a lot



#18 mdfenko

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Posted 12 November 2013 - 04:51 AM

you could use buffered urea or urea made up in water. sds and/or thiourea should not be necessary.

 

however, you could also add urea to the laemmli sample buffer. if you need to determine the protein concentration then you can omit the bromphenol blue from the sample buffer (add it just before loading) and do the protein determination using a method which is insensitive (or less sensitive) to the sds and reducing agent (you can also omit the reducing agent and add it prior to loading, if necessary).


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#19 Vemovied

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Posted 12 November 2013 - 05:30 AM

you could use buffered urea or urea made up in water. sds and/or thiourea should not be necessary.

 

however, you could also add urea to the laemmli sample buffer. if you need to determine the protein concentration then you can omit the bromphenol blue from the sample buffer (add it just before loading) and do the protein determination using a method which is insensitive (or less sensitive) to the sds and reducing agent (you can also omit the reducing agent and add it prior to loading, if necessary).

PERFECT!!!

I´m going to make a Laemly wo BrFBlue and bMercapto :)

Thanks once again!






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