Western Blotting Protocol
Reagents:
Homogenizing Buffer (500 ml)
1190 mg HEPES (pH 7.4)
93 mg EDTA (500 ul of .5M EDTA)
1 Tablet of Protease Inhibitors
Sterile Filter w/vac
1.5 M Tris (pH 8) (150 ml)
27.23 g Tris Base
80 ml H20
Adjust pH to 8.8 w/HCL
Bring total up to 150 ml with H20
Autoclave
Store at 4 C
0/5 M Tris (pH 6.8)
6 g Tris Base
60 ml H20
Adjust pH to 6
8 w/HCl
Bring total up to 100 ml w/H20
Autoclave
Store at 4 C
10X Running Buffer (pH 8.3)
30.3 g Tris Base
144 g Glycine
10 g SDS (10 ml of 10% SDS)
500 ml H20
Dissolve
Bring up to 1000 ml w/H20
10X Tris Glycine Stock
30.3 g Tris Base
144 g Glycine
1000 ml H20
6X Sample Buffer/Loading Buffer
7 ml 0.5 M Tris (pH 6.8)
2.6 ml Glycerol
1 g DTT
60 ul of 10% Brom Blue
400 ul 10% SDS
PBS-T
50 mls 1x PBS
200 ul Tween
PBS-T + Nonfat Dry Milk
50 mls 1x PBS
200 ul Tween
25 g NDM
10% Sodium Azide
1 g Sodium Azide
10 mls H20
Transfer Buffer
100 ml 10x Tris Glycine Stock
70 ml 7% Methanol
7 ml 10% SDS
Bring up to 1000ml with dH2O
Stacking Gel (4% Acrylamide)
6.1 ml H20
1.3 ml Acrylamide
2.5 ml Tris-HCl .5M (pH 6.8)
- ml 10% SDS
100 ul APS
10 ul TEMED
Resolving Gel (7.5% Acrylamide)
4.9 ml H20
2.5 ml Acrylamide
2.5 ml 1.5 M Tris HCl (pH 8.8)
- ml 10% SDS
100 ul APS
10 ul TEMED
10% APS
100 mg Ammonium persulfate
1 ml H20
Preparation of Tissue:
With Razor remove appropriate area of tissue
Place in 1ml of HB (w/PI)
Homoginize tissue in Polytron
With Pasteur Pipette suck up contents and place in a 2 ml eppendorf tube
Spin samples at 4 C (highest speed) 25 minutes
Save supernatant!
Remove 60 ul of Sup and add to 10 ul Sample Buffer (to run on gel, can also freeze here)
Remove 50 ul of Sup for BSA assay
Freeze the remainder of the Supernatant at –80C.
Quantify amount of protein with BSA Assay
Load Gel:
Load 7 – 10 ul of Rainbow marker
Load appropriate amount of Sample (in Sample Buffer)
Run at 120 V for 1 hr. (V= Const.)
Remove glass plates from Electrode Assembly and set aside
Place PVDF membranes in tray containing 100% methanol then immediately into tray of transfer buffer.
Place sponges and sandwich in T. Buffer as well
With spatula pry off top glass plate
With razor slice edges of gel (where it meets spacers)
Place dry whatmann paper on top of gel and peel off
Flip over so gel is facing up
Lay PVDF membrane smooth side down on top of gel
Make sandwich:
White – Sponge- Paper – PVDF – Gel – Paper – Sponge – Black
Place in sandwiches in container and fill up entire container with T. Buffer
Add Ice tray to container
Run 1.5 hrs at 120 V
Remove PVDF and place in glass tubes lined with Silk
Block with 10 ml PBS-T + NDM (15 min at RT) in rotating rack
Dump out PBST+NDM, and add in primary Ab Solution.
Primary Ab Solution
5-10 mls PBS-T with 5% milk or 3% BSA
Primary Ab 1:500-1000
0.05% Sodium Azide (0.005g in 10 ml)
Incubate at RT for 1 hour or at 4C overnight (1/2 Speed)
Day 2
Wash 3X 5min PBS-T (High Speed)
Incubate with Secondary Ab at 1:2000-4000
10 mls PBS-T + NDM
2.5 ul Secondary Ab
Incubate for 30 min at RT (High Speed)
Wash 3X 10 min PBS-T
ECL Detection:
Place PVDF in yellow pipette tip box
Add :
1000ul Solution A
1000ul Solution B
Right on top of membrane
Put on Rotator 2min
Sandwich PVDF between 2 sheets of plastic wrap
Put in small cassette
Place MR film on top (dark room) Dull side down
5 minute exposure
30 minute exposure
After developing film put film on top of membrane and mark where the rainbow markers are.
