User:Torsten Waldminghaus/protein gel western

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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
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