Western Blot

 

This protocol is adapted from the one provided by Bio-Rad, one of the manufacturers of the transfer sandwich used for a Western Blot. It should be applicable across brands, but it is worth checking out the protocol provided by your manufacturer.

If you manage to DIY all the pieces required for this protocol, please send me pics and info via the contact form. I’d love to feature your work!

Equipment & Consumables:

  • Cell Lysis Equipment & Consumables

  • SDS-PAGE Equipment & Consumables

  • Micropipette and sterile tips

  • Ice in Ice Bucket

  • Water Jet-spray Bottle

  • 1 x Transfer Buffer

    • 25 mM Tris Base

    • 190 mM Glycine

    • 20% methanol

    • 0.1% SDS for proteins > 80 kDa

  • Ponceau S Staining Buffer

    • 0.2% w/v Ponceau S

    • 5% glacial acetic acid

  • TBST Buffer

    • 20 mM Tris, pH 7.5

    • 150 mM NaCl

    • 0.1% Tween 20

  • Blocking Buffer

    • 3% Bovine Serum Albumin added to TBST buffer

  • Stripping Buffer

    • 20 ml 10% SDS

    • 12.5 ml 0.5 M Tris-Cl, pH 6.8

    • 0.8 ml β-mercaptoethanol

    • 67.5 ml dH2O

  • Primary Antibody

  • HRP-conjugated Secondary Antibody

  • Additional Primary/Secondary antibody pairs if you wish to search for additional proteins/include a control.

  • Chemiluminescent substrate appropriate for Secondary antibody

  • Appropriate illumination source for the substrate

  • Digital Camera

  • 2 x large plastic/glass tray large enough to hold 4 SDS-PAGE gels side by side.

  • Western Blot Transfer Sandwich

    • 2 x Filter Pads

    • 2 x Whatman Filter Squares

    • 1 x Transfer Membrane

  • Western Blot Cassette and Tank

    • Vice-like cassette for holding the gel

    • Tank for holding the running buffer.

    • One of these two will likely have the positive and negative electrode

    • Ice Block (this part will need to be frozen beforehand)

  • DC Power Supply and red/black wires

  • Cold room or a lot of cold packs + esky.

  • Rocker

Protocol:

DAy 1: Cell Lysis, SDS-PAGE, Membrane Transfer

  1. Perform a cell lysis protocol to lyse your cells.

  2. Load the protein extracts on an SDS-PAGE gel, run at 100V for ~1 hour.

    • You can photograph your gel with white light before continuing but you risk tearing it.

  3. Fill the plastic trays with 1 x Transfer buffer to a depth of 1-2 cm, then gently push the SDS-PAGE gel from its cassette into the tray using water.

    • An excellent technique for moving an SDS-PAGE gel without tearing it is to use a wash bottle to push it along with a jet of water. Trying to manually move it with your hands will nearly always end in disaster.

  4. Assemble your transfer sandwich, carefully ensuring there are no air bubbles between any layers. Start by placing the first fiber pad into the tray of transfer buffer, pushing it right to the bottom. Next, gently push the first piece of whatman paper down onto the fiber pad, smoothing out any air bubbles. Next move your SDS-PAGE Gel across under the water until it is lying square on top of the filter paper. Soak the transfer membrane next, then move it into position above the gel and smooth it down. Soak a second filter paper square, smoothing it over the membrane, then push the second thick fiber pad into position.

  5. The final product should look something like this (top to bottom - ie. reverse order from how you add them);

    • Thick Fiber Pad

    • Filter paper (Whatman paper)

    • Nitrocellulose Transfer Membrane

    • SDS-PAGE Gel

    • Filter Paper (Whatman paper)

    • Thick Fiber Pad

  6. Lift up your transfer sandwich by squeezing the fiber pads on either side, then move the entire sandwich to the cartridge/cassette that will hold it for electrophoretic transfer.

  7. Place the cassette and the ice block into the tank together, then put on the lid. The side of the sandwich with the nitrocellulose membrane should be closest to the red! Run towards red!

  8. If you have a cold room, move everything there now.

    • If you don’t have a cold room, you can place the tank in an esky filled with cold packs with the wires leading out.

  9. Run at 10 mA overnight in the cold room.

    • Optional: Or 100 V for 1-2 hours, depending on protein size.

Day 2: Blot Incubation with Primary antibody probes

  1. Disassemble the sandwich in a tray of dH2O and remove everything except for the blot.

  2. Pour off the water and submerge the blot in Ponceau S staining buffer within the cleaned plastic tray, incubate 5-10 minutes on a rocker (or manually shake back and forth).

  3. Pour off the Ponceau S staining buffer, wash 3 x 5 minutes with TBST buffer. Pour off the TBST buffer.

  4. Add Blocking Buffer (BSA in TBST) and block for 1 hour at RT.

  5. Dilute Primary antibody in the blocking buffer according to the manufacturer’s ratio.

  6. Incubate the blot overnight submerged in the Primary antibody solution in the cold room (or on ice blocks).

Day 3: Blot incubation with secondary antibody Probes & Imaging

  1. Rinse the blot 3 x 5 minutes with TBST buffer. Pour off the TBST buffer.

  2. Dilute HRP-conjugated Secondary antibody solution in TBST according to the manufacturer’s instructions.

  3. Incubate the blot in the Secondary antibody solution for 1 hour at RT.

  4. Rinse the blot 3 x 5 minutes with TBST buffer.

  5. Stain the blot with a chemiluminescent substrate (e.g. luminol based)

  6. Image the blot with a digital camera using illumination appropriate to the chosen chemiluminescent substrate.

    • I will need to write a dedicated article on this soon.

Optional: Stripping and Reprobing - If you want to check for additional proteins on the gel.

  1. Warm the stripping buffer to 50°C.

  2. Using a different tray, submerge the membrane in the stripping buffer. Incubate 45 mins at 50°C. Shake gently on the rocker if possible, manually otherwise.

  3. Rinse the blot 10 x 5 minutes with sterile water.

    • Otherwise find a way to run sterile water continuously over the blot.

  4. Transfer to a fresh tray, 5 x 5 min washes with TBST. Pour off the last wash.

  5. Add Blocking buffer and incubate for 1 hour.

  6. Incubate in the new Primary antibody solution at 4°C overnight.

    • This new antibody may bind to a control protein, or a secondary protein of interest.

  7. The next morning, rinse the membrane 3 x 5 minutes with TBST. Pour off the last wash.

  8. Add the new HRP-conjugated Secondary antibody solution, incubate for 1 hour at RT.

  9. Rinse the membrane 3 x 5 minutes with TBST buffer .

  10. Stain the blot with a chemiluminescent substrate (e.g. luminol based)

  11. Image the blot with a digital camera using illumination appropriate to the chosen chemiluminescent substrate.