Western Blot: wet blot

Materials
Western Transfer Buffer	dissolve 6.0 g Tris Base; 28.8 g glycine in 1 L dH2O add 430 mL methanol and degas for 30 min while stirring after degasing, add 10 mL 20% SDS and fill up to 2 L with H2O
Blocking Solution	TBS-T; 2% BSA; 0.05% NaN3
TBS-Tween	200 mL 10x TBS; 10 mL Tween; ad 2 L H2O
	First and second Antibody
	ECL-Medium
	H2O2 (30%)
Staining Solution	250 mL isopropanol; 100 mL acetic acid; 650 mL H2O; 0.03% Coomassie
Destaining Solution	10% (v/v) isopropanol, 10% (v/v) acetic acid

Procedure

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• The PVDF membrane is labeled with a pen and quickly activated in methanol, then soaked in Western transfer buffer.

  

  Schematic structure of a WB sandwich.

• The transfer chamber is assembled according to the instructions of the supervisor: The Western Blotting stack is assembled by placing a Wet Blotting clamp with the red side down in a plastic container filled with Western Transfer buffer. Place a foam pad followed by two Whatman papers on the red plastic cover. Place the PVDF membrane on the stack and put the PAA gel on top of the membrane. Place two Whatman papers on the gel and remove any bubbles trapped between the layers using a roller Top the stack with a black foam pad and close the Wet Blotting clamp and place it with the red plastic cover towards the anode (+) in the transfer chamber.

• The blot is connected to a power supply and the proteins are transfered over night at room temperature at 30V or for 2h at 200mA with cooling.

• Upon completion of transfer, the apparatus is disassembled and the membrane is further processed as described in “Western Blot: probing of the membrane”.