Please cite this paper if you use this method and look there for more detailed information!
Protocol
Steps of the staining procedure
Run protein extracts containing urease on a native PAGE gel
(for most samples from plant 10-20 mM DTT should be present in the protein extracts)
Equilibrate gel 5 times for 10 min in 5 microM sodium acetate buffer (100 ml per mini-gel)
(Conveniently a 5 mM sodium acetate buffer, pH 6.0 may be used as stock)
Equilibrate gel for 10 min in water
Place in staining solution (in sealed plastic bag)
Incubate at 37°C without shaking until bands are visible
(5 min to several hours)
Stop staining by repeated short incubations in water or 20 mM HCl
How to make staining solution
This gives 20 ml staining solution (enough for two mini-gels)
compound
stock concentration
final concentration
volume to add (ml)
urea
5 M
0.1 mM
0.4
NBT
0.5 % (w/v)
0.08 % (w/v)
3.2
DTT
50 mM
0.5 mM
0.2
H2O
--
--
16
DTT = dithiothreitol
NBT = nitroblue tetrazolium
(0.5 % solution is stable in the frige for several month at least)
