Western Blotting: Example protocol

In the following you may find an example of a Western Blotting protocol. All values are merely examples and must be adjusted by the experimenter to the equipment and individual demands. Please observe that each antibody requires adjustment to your individual experimental set-up. Especially concentrations and incubation-times must be adjusted and optimized individually.

1. Prepare solutions and buffers

A. Buffer preparation

Resolving Buffer Store at 4°C)
90,9 g Tris (1,5 M)
500 ml dest. H2O
pH 8,8 adjusted

Stacking Buffer (Store at 4°C)
30,2 g Tris (0,5 M)
500 ml dest. H2O
pH 6,8 adjusted

10x Running Buffer (Store at Room temperature (RT))
30 g Tris
144 g Glycin
10 g SDS
1 l dest. H2O
pH 8,6 adjusted; discard in case of large difference

Transfer Buffer (Store at 4°C):
6 g Tris (25 mM)
28,8 g Glycin (190 mM)
400 ml Methanol (20%)
2000 ml dest. H2O

Tris-Tween Buffered Saline

10X TTBS:	1x TTBS equals:	Needed for 2 l:
87,66 g NaCl (1,5 M) 39,4 g Tris-HCl (250 mM) 20 ml Tween 20 (0,01%) Ad 1000 ml Aqua dest pH 7,4	50 mM Tris pH8.0 150 mM NaCl 0,1% Tween20	50 ml of 1 M Tris, pH 8.0 Stock-solution 60 ml of 5 M NaCl Stock 8 ml of 25% Tween20 Stock

Dried milk (for 100 ml):
3-5 g skimmed milk-powder (non fat!)
100 ml TTBS

BSA (for100 ml):
2,5-5 g BSA
100ml TTBS

2x Sample Buffer (reducing)
2,4 ml Aqua dest
2,4 ml Stacking buffer
2,0 ml Glycerol
2,0 ml SDS (10%)
200 µl Bromphenolblue
40 µl EDTA (0.5 M)
1,0 ml ß-Mercaptoethanole

Add ß-Mercaptoethanole just before usage
Sample Buffer with Mercaptoethanol can be used for 2-3 weeks

B Gelmix

Separation-gel

Number of gels	4	4	2	4	4
Concentration	5%	7,5%	10,0%	10,0%	12,5%
Volume (total) H2O Resolving Buffer SDS 10% Acryl-/Bisacrylamide 30% TEMED APS 10%	ca. 25 ml 14,3 ml 6,3 ml 250 µl 4,2 ml 12,5 µl 125 µl	ca. 25 ml 9,6 ml 5 ml 200 µl 5 ml 20 µl 200 µl	ca. 10 ml 4 ml 2,5 ml 100 µl 3,4 ml 10 µl 100 µl	ca. 25 ml 8 ml 5 ml 200 µl 6,8 ml 20 µl 200 µl	ca. 25 ml 6,6 ml 3,8 ml 200 µl 8 ml 20 µl 200 µl

Stacking-gel

Number of gels	1	2	4
H2O Stacking Buffer SDS 10% Acryl-/Bisacrylamid 30% TEMED APS 10%	1,2 ml 0,5 ml 20 µl 260 µl 2 µl 10 µl	2,4 ml 1 ml 40 µl 520 µl 5 µl 50 µl	4,8 ml 2 ml 80 µl 1040 µl 10 µl 100 µl

2. Elektrophoresis

- Assemble Western Blotting equipment for gel casting (top off with a small amount of isopropanole)
- After 15 min discard Isopropanole
- Cast stacking-gel on top
- Add combs, wait ca. 15 min
- In the meantime: prepare samples

- x µl sample plus x µl sample buffer
- 3-5 min at 5°C; then centrifuge, keep on ice

- Add gel to electrophoresis-chamber
- Add 1x Running Buffer (chilled previously)
- Remove combs
- Load samples and standards
- Elektrophoresis:

100 V ca. 15-20 min (until samples arrive in separation-gel) RT
150 V ca. 1 h RT

3. Blotting

- Wash gels in transfer-buffer
- Cut membrane into 9x6 cm large pieces
- Soak sponges, paper and membranes thoroughly (in transfer buffer)
- Assemble blotting-sandwich:

- Order: Holder-Sponge-Paper-Gel-Membrane-Paper-Sponge-Holder

Observe: bubbles need to be removed!

- Add sandwich to chamber- make sure running-direction is correct!
- Add block of ice
- Fill chamber with 1x transfer-buffer
- Transfer:

100 V 1h RT or. 50 V 2h
10 V over night

4. Blocking

- Wash membrane 3x (in TTBS)
- Block in 2,5-5% BSA in TTBS (alternatively in blocking milk) 1h RT/37°C
- 3x 10 min washing in TTBS

5. Primary antibody

- Dilute in TTBS e.g. 1:1000
- Over night 4°C or a few hours at RT (observe instructions on datasheet)
- Collect antibody dilution and store at 4°C

6. Secondary antibody

- Wash in TTBS thoroughly (at least 3x)
- Dilution (antibody) in TTBS e.g. 1:5000
- 1,5-2h at RT
- Wash in TTBS thoroughly, 2h

7. Develop with ECL

- Put membrane on a foil
- Combine ECL solutions

- mix
- add to membrane
- allow time to react
- drain

- Add film in dark-chamber and develop