TITLE: Western blotting using the BioRad Criterion Blotter system
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for performing an SDS-PAGE Western blot analysis using the BioRad Criterion Blotter system.

[STEPS]
SECTION: Day 1
1. Load samples onto a 4-12% Bis-Tris (NuPage) gel with MOPS buffer (ThermoFisher). If using SDS loading dye (with 5% SDS final concentration), all lanes must contain the same volume of sample. Ensure all lanes are filled with 1x SDS loading dye at the same volumes as the sample/marker lanes. If this is not done, samples will spread according to the total sample volume, and the wells will not run evenly. 
SECTION: Day 1
2. Run gels according to the following gradient: 100 V (10 min), 150 V (10 min), 200 V (until the dye front has reached the desired distance – typically ~40 min). 

SECTION: Day 1
3. Cut 1x piece of PVDF (0.45 um), and 2x pieces of Whatman paper per gel, according to the gel dimensions. 
SECTION: Day 1
4. Place the PVDF membrane into methanol for ~ 5 seconds, remove the methanol and wash the membrane three times in distilled water.
SECTION: Day 1
5. Place the membrane in cold ( 4 °C ) 20% methanol Bis-Tris transfer buffer (Thermofisher)

SECTION: Day 1
6. Remove the gel from the cassette and place in ice cold 20% methanol Bis-Tris transfer buffer (ThermoFisher)
SECTION: Day 1
7. Add a frozen ice pack to the Criterion Blotter transfer tank, and fill the tank with cold transfer buffer.
SECTION: Day 1
8. Soaking each Whatman paper piece in transfer buffer for ~ 5 sec before use, assemble the following transfer stack: 1x Whatman, SDS-PAGE gel, PVDF membrane, 1x Whatman, rolling each layer to prevent bubbles accumulating between pieces
SECTION: Day 1
9. Place the transfer stack between sponges and secure them in the cassette in the correct orientation, and place the cassette in the transfer tank. If transferring 1 gel, ensure an empty cassette containing only sponges is placed in the second cassette holder in the tank. 
SECTION: Day 1
10. Run the transfer at 100 V for 60 min. 

SECTION: Day 1
11. Remove the membrane from the transfer stack and place into destain (50% methanol/7% acetic acid) for 2 min rocking at Room temperature. 

SECTION: Day 1
12. Wash the membrane 3x in 0.05% v/v Tween/PBS for 2 min rocking at Room temperature. 

SECTION: Day 1
13. Incubate the membrane in 3% w/v milk powder in 0.05% v/v Tween/PBS for 20 min rocking at Room temperature. 

SECTION: Day 1
14. Wash the membrane 3x in 0.05% v/v Tween/PBS for 3 min rocking at Room temperature. 

SECTION: Day 1
15. Place the membrane in the desired primary antibodies made up in 3% w/v BSA in 0.05% v/v Tween/PBS and incubate rocking, either overnight at 4 °C or for 60 min at Room temperature. 

SECTION: Day 2
16. Wash the membrane 3x in 0.05% v/v Tween/PBS for 3 min rocking at Room temperature. 

SECTION: Day 2
17. Place the membrane in the desired secondary antibody in 3% w/v milk powder in 0.05% v/v Tween/PBS for 60 min rocking at Room temperature.

SECTION: Day 2
18. Wash the membrane 2x in 0.05% v/v Tween/PBS for 3 min rocking at Room temperature. 

SECTION: Day 2
19. Wash the membrane once in PBS for 3 min rocking at Room temperature. 

SECTION: Day 2
20. Incubate the membrane in ECL developing reagent for 5 min, prior to developing the blot using a ChemiDoc Imaging System.