Western Blot: semi-dry blot: Difference between revisions
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Created page with "{| class="wikitable" |+ !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, 10 ml MeOH, fill up to 100 ml with ddH2O * Cathode Buffer preparation: 20 ml 5x Cathode buffer, 10 ml MeOH, fill up to 100 ml with ddH2O * The PVDF membrane is labeled with a pe..." |
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* Anode Buffer preparation: 20 ml 5x Anode buffer, | * 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, | * 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 | * 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 | * 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 | ||
Latest revision as of 13:11, 16 June 2025
| 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”