Western Blot (Protein Blotting onto a PVDF Membrane, Traditional Method)
Introduction
Materials
- PVFD (Polyvinylidene fluoride) Membrane
- Bio-Rad Mini Trans-Blot Module
- Bio-Rad Trans-Blot Mini Tank
- Filter Paper
- Fiber Pads
- Forceps
- Blot Roller
- 1X SDS-Page Transfer Buffer
- TBS-T
- Blocking Solution (TBS-T at 1% BSA)
- Optionals if staining:
- Ponceau-S Staining Solution
Procedure
Activate the membrane and equilibrate the gel
- Note: Place the Bio-Ice cooling in your laboratory freezer at -20°C until ready to use. After use, return the cooling unit to the freezer for storage.
Important: Current to the cell, provided from the external power supply, enters the unit through the lid assembly, providing a safety interlock to the user. The current to the cell is broken when the lid is removed. Do not attempt to circumvent this safety interlock, and always turn the power supply off before removing the lid, or when working with the cell in any way.
- Chill (4°C) transfer buffer because this will improve heat dissipation during transfer.
- Cut PVDF (Polyvinylidene fluoride) membrane (per gel) and filter papers (two per gel) to the dimensions of the gel. Always wear gloves when handling membranes to prevent contamination.
- Cut a little bit of the lower right corner of the membrane to always visualize the membrane in the proper orientation (in other words in the order that were loaded the samples in the gel).
- CRITICALActivate PVDF membrane by washing it in 100% methanol before equilibrating in transfer buffer.
- Use transfer buffer to equilibrate the gel
Note: Equilibration time fro gel will depend on gel thickness.
- Wash membrane with Running Buffer
- Pre-wet the filter paper and fiber pads with running buffer
Prepare the gel-filter-fiber sandwich.
- Place the transfer cassette open, with the black side down, on a clean plastic container with transfer buffer.
- Place one pre-wetted fiber pad on the black side of the cassette.
- Press a pre-wetted sheet of filter paper onto the equilibrated gel to fetch the gel from the container
Make sure that lane 1 (marker) is on your right side and the corner that was cut on the left side of the filter paper.
- Place the pre-wetted membrane on the gel aligning the lower cut of the membrane with the lower cut of the gel (this will ensure that when you remove the membrane you will have the marker on the left side of the membrane and your samples on the right).
- CRITICALUse a blot roller to gently press out any air bubbles between the PVDF membrane and the gel.
- Complete the sandwich by placing a piece of pre-wetted filter paper on the membrane. Use a glass tube or the blot roller to gently roll air bubbles out from between the membrane and the filter papers.
- Add the last pre-wetted fiber pad.
- Close the cassette firmly, being careful not to move the gel and filter paper sandwich.
- Lock the cassette closed with the white latch.
- Repeat for the other cassette if necessary for another gel.
Preparing the Transfer Tank
- Place the cassette in the transfer tank.
- Add the frozen Bio-Ice cooling unit to the side of the transfer tank
- Completely fill the tank with chilled transfer buffer.
- Add a standard stir bar to help maintain an even buffer temperature and ion distribution in the tank. Set the speed as fast as possible to keep ion distribution even.
- Put on the lid, plug the cables into the power supply, and run the blot at constant Voltage, 100V, 0.35 A for 1 hour.
- Upon completion of the run, disassemble the blotting sandwich and remove the membrane for follow-up staining or immunodetection.
Membrane Staining (Optional)
Note: Staining is optional. Membranes can be directly used with immunodection protocol without being stianed.
- Place your membrane inside a plastic container with enough Ponceau-S Staining Solution. Stain for 5 minutes.
- Visualize the proteins bands of your samples.
- Rinse membrane with TBS-T until the background is completely clear.
- If you are going to use the membrane immediately leave the membrane in a plastic container with TBS-T
Membrane Storage
Membranes are usually stored for later use. Conditions will depend on the intended storage time.
- Rinse the membrane with blocking solution for 1 hour at 4ºC
- Store membrane in a sealed container with TBS-T.
short period (< 1 week): Store membrane at 4°C.
long period (> 1 week): Store membrane at -20°C.
NOTE: Do not let the membrane dry at room temperature. Keeping the membrane moist during the procedure is crucial for obtaining reliable and reproducible results. Drying the membrane can lead to protein denaturation and irreversible structural changes. This can result in the loss of antigenic epitopes and decreased antibody binding efficiency during subsequent steps of the Western blotting process. Dry membranes tend to have higher levels of non-specific binding, leading to higher background noise and reduced signal-to-noise ratio. Keeping the membrane moist helps to minimize non-specific binding and improves the overall quality of the Western blot.
Materials Disposal
- Transfer buffer disposal
Use the transfer buffer up to 3 times and then transfer to an
appropriate container for disposal through the University’s Chemical Waste Program.
- Polyacrylamide Gel Disposal
Although polymerized acrylamide (non toxic) is not regulated as a hazardous waste, polyacrylamide gels often contain un-polymerized acrylamide which is a toxic material that can produce a hazard when introduced to the environment.
Use the following guidelines when disposing of polyacrylamide gels.
- Polyacrylamide gels should be disposed through the University’s Chemical Waste Program.
NOTE: Do not dispose of polyacrylamide gels in the regular trash or in red bags as a biological waste.
- Polyacrylamide gels should be placed into a leak- proof bag. Seal the bag and place the sealed bag inside a cardboard box. Do not use red biological waste bags or any type of bag or box marked with the biohazard symbol.
- A completed “WASTE CHEMICALS” label should be affixed to the box.
- Identify the waste as “polyacrylamide gel” and process through the Chemical Waste Program.
- Gloves and debris visibly contaminated with polyacrylamide gels should be placed in a separate sealed plastic bag. Place the sealed bag inside a cardboard box and label as above. Do not use red biological waste bags or any type of bag or box marked with the biohazard symbol. Dispose through the Chemical Waste Program.
Protocol for transferring (blotting) proteins into a membrane.
Authors: Osvaldo D. Rivera-Gonzalez, PhD & Luis Vazquez-Quiñones, PhD
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