11-10-2001
Western Blots from cochlea
1. Harvest the tissue in cold PBS or HBSS or DMEM, 2 at a time.
try 10 cochleas.
For the fine dissection in the sylgard dish, add some protease inhibitors to the
mix.
Mireille's cocktail –
a. 10µg/ml leupeptin.
b. 10µg/ml pepstatin.
c. 10µg/ml aprotinin.
d. 1mg/ml AEBSF.
e. (5mM EDTA and 50mM NaF to study phosphorylation of proteins).
2. Transfer the dissected tissue (with a pipette) to an eppendorf tube containing the
same dissecting buffer with protease inhibitors, on crushed ice.
3. Spin the tube at 40C for 1min at 12,000rpm.
4. Remove supernatant and add lysis buffer:
a. 2% SDS.
b. 10% glycerol.
c. 62.5mM Tris-HCl pH 6.8.
lysis buffer can be stored at -200C.
(for P0 rat thermolysin rat utricle Mireille used 50-80µl of buffer for 1218 utricles – yielding a 1µg/µl – and loaded 10-20µl per blot which were
10-20µg).
I'll add 100µl of lysis buffer for 10cochleas.
5. Crush the tissue using a blue pester fit for an eppendorf tube or do the entire
process in the small glass homogenizer.
6. Sonicate for 10 seconds on crushed ice.
7. Bradford assay the homogenate.
8. Add 5% volume of β-mercapto-ethanol (the other components of the loading
buffer are covered for – see table at the end).
9. Heat at 95ºC for 5 min.
At this point you can store at –80C.
Always thaw on crushed ice, or at 4C. You can thaw multiple times, or make
aliquots.
According to the Bradford assay results the homogenate can be further diluted. A
final protein concentration of 2µg/µl is considered very good, (when mounting
11-10-2001
10-20µl per lane).
10. For westerns – use the myosin VIIa Tamma Hasson antibody at 1µg/ml
(according to her technical sheet).
Gel selection (from the biorad site)
Tris-HCl gel
MW resolved (kD)
5%
100-250
7.5%
40-200
10%
30-150
12%
20-120
15%
10-100
18%
6-50
4-15%
20-250
4-20%
10-200
10-20%
10-100
Myosin VIIa
Myosin VI
Pou4F3
254, 847 Dalton
146,408 Dalton
~42,000 Dalton
Lysis buffer:
2% SDS.
10% glycerol.
62.5mM Tris-HCl pH 6.8.
Sample buffer 2X (Mireille):
0.5M Tris-HCl pH 6.8
10% SDS
0.1% bromophenol blue
Glycerol
Β-mercaptoethanol
Water to
2.5ml
4ml
0.5ml
2ml
0.5ml
10ml
Final concentration in sample
50mM
2%
0.0025%
10%
2.5%
The lysis buffer covers for all of the components of the sample buffer (but the
bromophenol blue that we can do without and the β-mercaptoethanol that should be
added after the BCA assay as it interferes with it).