User:Torsten Waldminghaus/protein gel western

Recipe for buffers

Resuspension buffer

20 ml TE buffer

5 ml Stacking gel buffer

3.5 ml 10% SDS

1.5 ml glycerol

4 ml ddH2O

• Store at rt.

Loading buffer

285 μL 2.5M DTT

205 μL 10%SDS

10 μL 10% Bromphenolblue

• Store at -20°C
• Should be 15% of endvolume (for example 30 μL sample + 5.3 μL Loading buffer

Western

• activate the membrane (for example Immobilin -P, Millipore) with methanole and then equilibrate with water.

Semi dry blot assembly

1. 2 filter papers with Anode buffer I
2. 1 filter papers with Anode buffer II
3. Membrane
4. Gel
5. 3 filter papers with Cathode buffer

• Run 30 min at 15V

Anode buffer I

36.3 g Tris

200 ml methanol

H2O to 1 liter

Anode buffer II

3.03 g Tris

200 ml methanol

H2O to 1 liter

Cathode buffer

5.2 g 6-amino-n-hexanoic acid

200 ml methanol

H2O to 1 liter