Runing problems - (Oct/24/2013 )

mdfenko on Tue Oct 29 11:30:54 2013 said:

wash the whole extract, before adding laemmli buffer.

 

make sure you remove all of the organic phase or, at least, that you don't carry any over into the gel sample.

Sorry for my question, but when you use a organic wash, I was thinking that my protein will go to the organic (chloroform) phase, isn´t  it?

no. most proteins will remain in the aqueous phase.

mdfenko on Wed Oct 30 12:06:13 2013 said:

no. most proteins will remain in the aqueous phase.

Thanks a lot

I will try it just now!!!

Thanks!!

phage434 on Mon Oct 28 23:54:59 2013 said:

You could extract the fat with a chloroform wash.

It was fantastic, the fat was removed completely, but I´m in trouble now for re suspend the protein, any suggestion for a good buffer?

Thanks so much!

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.

phage434 on Mon Nov 11 16:18:10 2013 said:

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!

phage434 on Mon Nov 11 16:18:10 2013 said:

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

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).

mdfenko on Tue Nov 12 12:51:29 2013 said:

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!