Team:TU Darmstadt/Protocols/Western Blot

Western Blot

Materials

Equipment

• Blot chamber (Transblot SD Semi Dry transfer cell)
• plastic bowl
• SDS gel

Chemicals & consumables

• 3% milk powder PBS buffer pH=7.4
• 1.5% milk powder PBS buffer pH=7.4
• 0.05% Tween 20 in PBS pH=7.4
• 1. Antibody (mouse-antimyc)
• 2. antibody (goat-antimouse-AP)
• NBT
• BCIP
• Blotting buffer
• AP buffer
• nitrocellulose membrane (Whatman Nitrocellulose membran)
• filter paper

Procedure

Transfer

1. seperate proteins via SDS gel (SDS PAGE)
2. put 3 layers of filter paper (drenched in Blotting buffer) on the graphite diode of the apparatus
3. cover it with nitrocellulose membrane with the size of your SDS gel
4. place SDS gel on top of it (should be wet)
5. cover it with 3 layers of filter paper (drenched in Blotting buffer)
6. put cathode on top of the apparatus
7. transfer of from SDS gel to membrane is performed by adding a constant current of 12 V for 1 h
8. afterwards the nitrocellulose membrane, carrying the proteins, is blocked by incubating it in 3% milk powder PBS buffer for 2 h at room temperature or over night at 4°C

Staining

1. wash membrane with Tween PBS buffer 3x for 5 min each
2. add 1.5% milk powder PBS carrying 1. antibody in 1:1000 concentration

   incubate for at least one hour at RT, keep shaking the whole time

3. wash membrane with Tween PBS buffer 3x for 5 min each
4. add 1.5% milk powder PBS carrying 2. antibody in 1:10000 concentration

   incubate for at least one hour at RT, keep shaking the whole time

5. wash membrane with Tween PBS buffer 3x for 5 min each
6. wash membrane with ddH₂ once for 5 min
7. add AP buffer, 20 µL of NBT and 70 µL of BCIP
8. keep shaking for 1 h