ESO wikiadmin at 12:11, 16 June 2025

2025-06-16T12:11:13Z

← Older revision		Revision as of 14:11, 16 June 2025
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	* Anode Buffer preparation: 20 ml 5x Anode buffer, 10 ml MeOH, fill up to 100 ml with ddH2O		* 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, 10 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		* 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

ESO wikiadmin: 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..."

2025-02-27T13:26:48Z

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..."

New page

{| 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 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”

Western Blot: semi-dry blot - Revision history

ESO wikiadmin at 12:11, 16 June 2025