GR Western Blot in brain tissue
1. TEMDG buffer (50 ml):
Tris Base 60 mg
Molybdic Acid (disodium salt) 121 mg
EDTA 19 mg
Dissolve the above in 40 ml H2O and Place solution on ice
Adjust pH to 7.6 with diluted HCl
Add Dithiothreitol (DTT) 39 mg
Glycerol 5 ml
Leupeptin (1 mg/ml) 0.25 ml
Aprotinin (1 mg/ml) 0.5 ml
Trypsin inhibitor (10 mg/ml) 0.3 ml
PMSF (100 mM in ethanol) 0.5 ml
Check that pH is close to 7.6, otherwise correct
Add H2O to complete 50 ml, mix.
2. Dounce homogenization:
Add 0.5 ml TEMDG/100 mg frozen tissue to the glass homogenizer
(placed on ice-water mix),
Homogenize with 10 strokes with pestle A (the looser fit one)
Followed by 10 strokes with pestle B (the tighter fit one)
Centrifuge 2 h at 100,000 x g at 4 C degrees and take the supernatant
(cytosolic fraction)
3. SDS-PAGE (8% gel)
For each lane mix 30 ul cytosol with 10 ul 4X loading buffer (see
below); run a lane with MW standards. Boil samples in bath for 5 min.
Load gel and run until the dye front is about to fall.Transfer gel to PVDF membrane
4. For Western detection:
Block membrane 1h in 7% non-fat dry milk, 0.5 % Tween 20, 1 X TBS
Wash 3 times x 5 min in wash buffer: 1 X TBS, 0.1 % Tween 20
Incubate 1 h with BuGR2 (1:4000 dilution) in 1 X TBS, 0.1 % Tween 20, 1 % dry milk
(AB buffer)
Wash 3 times x 5 min in wash buffer
Add anti mouse Ig-peroxidase coupled antibody (1/4000 dilution in AB buffer)
Develop with ECL substrates 1/1 mix
5. GR should be detected close to the 97 KDa marker. If there is
degradation it is usually as a 45 KDa band.
6. 4 X Loading buffer (for 10 ml total after beta-mercaptoethanol addition):
0.5 M Tris-HCl (pH 6.8) 4 ml
SDS 0.8 g
Glycerol 4 ml
Bromophenol blue 10 mg
Add betamercaptoethanol to 20 % (V/V) just before use