3 replies to this topic

[hjc05]

Hi everyone,

I am having a problem with my nuclear extraction. I am looking for phosphorylated proteins in nuclear extracts from T84 cells. I use a high salt extraction procedure. Do I need to precipitate my proteins before running a western because of the high salt concentration? I tried TCA precipitation, but I did not get a good yield. Does anyone have a good recommendation? I resuspend my nuclear pellet in buffer B and then add an equal amount of buffer C.

Buffer B:
20 mM Hepes
1.5 mM MgCl2
420 mM NaCl
.2 mM EDTA
25% glycerol
.5 mM DTT
and protease and phosphatase inhibitors.

Buffer C:
20 mM Hepes
50 mM KCl
.2 mM EDTA
.5 mM DTT
and protease and phosphatase inhibitors.

Thanks for any help!

[Inmost sun]

You will probably get in trouble if using the non-desalted extract directly to a gel; a very good alternative to TCA is the precipitation method by of Wessel & Flügge (Anal Biochem, vol 138:141-143 (1984))

add to 100 µl aqueous extract

+400 µl methanol
+100 µl chloroform
+300 µl water

overhead shaking for 10 s

centrifuge 14,0000 g , 2 min

aspire the overlay aqueous phase, be careful, the precipitated proteins are the interphase

add 300 µl methanol

overhead shaking for 10 s

14,0000 g , 2 min

proteins are precipitaed

aspire the aqueous/methanol phase

dry to light humidity but not to fully dryness

Edited by Inmost sun, 14 July 2010 - 05:17 AM.

[laurequillo]

hjc05, on Jul 13 2010, 06:49 PM, said:

Hi everyone,

I am having a problem with my nuclear extraction. I am looking for phosphorylated proteins in nuclear extracts from T84 cells. I use a high salt extraction procedure. Do I need to precipitate my proteins before running a western because of the high salt concentration? I tried TCA precipitation, but I did not get a good yield. Does anyone have a good recommendation? I resuspend my nuclear pellet in buffer B and then add an equal amount of buffer C.

Buffer B:
20 mM Hepes
1.5 mM MgCl2
420 mM NaCl
.2 mM EDTA
25% glycerol
.5 mM DTT
and protease and phosphatase inhibitors.

Buffer C:
20 mM Hepes
50 mM KCl
.2 mM EDTA
.5 mM DTT
and protease and phosphatase inhibitors.

Thanks for any help!

The buffer I use for nuclear extraction is 400mM NaCl, 20mM Hepes pH 7.9, EDTA, EGTA, B-Mercaptoethanol and the inhibitors, and I never had any problem loading the samples directly.
Did you try to load your samples directly without purification?

Edited by laurequillo, 14 July 2010 - 07:08 AM.

[hjc05]

I had not tried loading my samples directly because they were too dilute. Now that I know what yield I'm getting, I'll use a smaller volume and try loading directly. Thanks for the help.