Western Blot Protocol
- Gel running apparatus
- Transfer apparatus
- Power supply
- SDS‐PAGE mini gel
- Nitrocellulose membrane cut to size of gel
- Filter Paper (Whatman) cut to size of gel
- X‐ray film
- X‐ray processor
- Gel loading pipette tips
- Pipettor, small volumes
- 25 mM Tris
- 192 mM Glycine
- 0.1% SDS
- 20 mM Tris
- 150 mM Glycine
- 20% methanol
- 0.038% SDS
- 5% non‐fat dry milk
Wash Buffer (TBST)
- 125 mM NaCl
- 25 mM Tris pH 8.0
- 0.1% Tween‐20
Running Protein Samples onto a Gel
- Cast a mini SDS‐PAGE gel per your labs standard protocols or purchase pre‐made gels.
- Clamp the gel to the apparatus with per manufacturer directions. Add running buffer.
- Remove the comb gently so as to not disturb the wells.
- Add 6μL of your select marker to a well.
- Add 7.5μL of lysate per well (2mg/mL for 15μg per lane).
- Apply the anode and cathode wires to the appropriate poles and cover.
- Run at 130V for 2 hours (or until the dye front is close to the bottom).
Transfer of Proteins onto Membrane
- Assemble the “sandwich” for the transfer apparatus per the diagram below.
- Note: Handle gel and membrane with tweezers – do not touch!
- Pre‐wet sponges and filter paper in transfer buffer.
- Note: Filter papers and membrane should be same size as the gel.
- Insert the “sandwich” and insert into the transfer apparatus, making sure the gel is on the cathode (‐) and the membrane is on the anode (+) side of the apparatus.
- Transfer the proteins to the membrane at 250mA in transfer buffer for 2 hours
- Remove the cassette from the apparatus, discard the gel, and place membrane on paper towels to dry. (Wash the membrane in 1xTBST to remove any leftover gel.)
- Incubate the blot with blocking buffer overnight at 4°C or 2 hours at room temperature with gentle agitation.
- Remove blot from blocking solution.
Primary Antibody Incubation
- Dilute antibody to the recommended dilution in 10mL of blocking buffer.
- Incubate the blot with the primary antibody for one hour at room temperature or overnight at 4°C.
- Wash the blot three (3) times 10 minutes each in washing buffer with gentle agitation.
Secondary Antibody Incubation
- Dilute 1μL anti‐rabbit IgG‐HRP conjugated secondary (or other appropriate secondary) in 10mL of blocking buffer to make a 1:10000 dilution
- Note: working dilution of secondary can vary from 1:2000 to 1:10000.
- Incubate blot with secondary antibody for one (1) hour at room temperature.
- Wash three (3) times for 10 minutes each in washing buffer with gentle agitation.
- Drain wash buffer
- Add ECL solution (Amersham) per manufacturer directions and develop for 1 minute.
- Drain the fluid.
- Cover the blot in plastic wrap.
- Expose the blot to X‐ray film for 1 minute in a dark room.
- If there is no banding, expose the film for 5 minutes, then 30 minutes and up to overnight if the signal is weak.
- If the signal is strong, expose the film for 30 seconds or less.
- Develop the film in an X‐ray processor.
- Optimal dilutions should be determined by each laboratory for each antibody.