Aug 09, 2023
Western blotting using the BioRad Criterion Blotter system
- 1Lazarou Lab, WEHI
Protocol Citation: Louise Uoselis 2023. Western blotting using the BioRad Criterion Blotter system. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkowy1v5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86225
Keywords: ASAPCRN
Abstract
Protocol for performing an SDS-PAGE Western blot analysis using the BioRad Criterion Blotter system.
Day 1
Day 1
1h 47m
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.
Run gels according to the following gradient: 100 V (00:10:00 ), 150 V (00:10:00 ), 200 V (until the dye front has reached the desired distance – typically ~40 min).
20m
Cut 1x piece of PVDF (0.45 um), and 2x pieces of Whatman paper per gel, according to the gel dimensions.
Place the PVDF membrane into methanol for ~ 5 seconds, remove the methanol and wash the membrane three times in distilled water.
Place the membrane in cold ( 4 °C ) 20% methanol Bis-Tris transfer buffer (Thermofisher)
Remove the gel from the cassette and place in ice cold 20% methanol Bis-Tris transfer buffer (ThermoFisher)
Add a frozen ice pack to the Criterion Blotter transfer tank, and fill the tank with cold transfer buffer.
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
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.
Run the transfer at 100 V for 01:00:00 .
1h
Remove the membrane from the transfer stack and place into destain (50% methanol/7% acetic acid) for 00:02:00 rocking at Room temperature .
2m
Wash the membrane 3x in 0.05% v/v Tween/PBS for 00:02:00 rocking at Room temperature .
2m
Incubate the membrane in 3% w/v milk powder in 0.05% v/v Tween/PBS for 00:20:00 rocking at Room temperature .
20m
Wash the membrane 3x in 0.05% v/v Tween/PBS for 00:03:00 rocking at Room temperature .
3m
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 01:00:00 at Room temperature .
1h
Day 2
Day 2
1h 14m
Wash the membrane 3x in 0.05% v/v Tween/PBS for 00:03:00 rocking at Room temperature .
3m
Place the membrane in the desired secondary antibody in 3% w/v milk powder in 0.05% v/v Tween/PBS for 01:00:00 rocking at Room temperature .
1h
Wash the membrane 2x in 0.05% v/v Tween/PBS for 00:03:00 rocking at Room temperature .
3m
Wash the membrane once in PBS for 00:03:00 rocking at Room temperature .
3m
Incubate the membrane in ECL developing reagent for 00:05:00 , prior to developing the blot using a ChemiDoc Imaging System.
5m