Western Blot: semi-dry blot
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| Materials | |
|---|---|
| 5X Anode buffer | 15 g Tris Base, pH to 10.4, final volume 1l |
| 5X Catode buffer | 15 g Tris Base, 26.25 g 6-amino-N-caproic acid (6-Amino-hexansäure), pH 9.4; final volume 1 l |
Procedure
- Anode Buffer preparation: 20 ml 5x Anode buffer, 20 ml MeOH, fill up to 100 ml with ddH2O
- Cathode Buffer preparation: 20 ml 5x Cathode buffer, 20 ml MeOH, fill up to 100 ml with ddH2O
- The PVDF membrane is labeled with a pen and quickly activated in methanol, then rinsed with water and finally soaked in Anode Buffer
- Assemble the semi-dry transfer chamber and build the “transfer sandwich”: Soak 3x Whatman filter paper in Anode buffer and place on anode place the membrane on the stack of soaked Whatman paper place the SDS-PAGE gel on top of the membrane
- Soak 3x Whatman filter paper in Cathode buffer and place on the gel.
- Important: remove air bubbles trapped between the sandwich layers and especially between the membrane and the SDS-gel by using a roller
- Put the Kathode on top of the assembly and plug in the transfer chamber into the power supply
- Run the transfer at 50 mA (as a rule of thumb use 1 mA per cm² of the gel/membrane) for ~1:30h (for smaller proteins) or ~2h (for larger proteins); the time as well as the mA can be optimized for your protein of interest
- Upon completion of transfer, the apparatus is disassembled and the membrane is further processed as described in “Western Blot: probing of the membrane”