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Western Blotting ProtocolDOWNLOAD A PDF
This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.
Sample Preparation- Lyse sample in the appropriate lysis buffer.
- Reduce and denature the sample by adding sample buffer and heat for 5 minutes at 95°C.
- Centrifuge at 16000 x g in a microcentrifuge for 5 minutes.
- Load 20-30μg of lysate per lane (or 100ng of purified protein) along with a molecular weight marker.
- Prepare the running buffer and assemble the gel in the tank.
- Drain blocking buffer from slide.
- Run SDS-PAGE for 30 minutes at an initial voltage of 75V for 20 minutes, then 115V for 60 minutes.
- Prepare transfer buffer.
- Cut a piece of membrane and wet in methanol or Tris-Buffered Saline + Tween (TBST) depending on membrane type. Transfer the membrane to 1x transfer buffer.
- Assemble transfer stack.
- Run transfer, 100V for 70 minutes.
- Check the transfer with Ponceau or Coomassie stain.
- Block the membrane by incubating for 1 hour in blocking buffer (TBST with 5% BSA or milk).
- Incubate the membrane with primary antibody in blocking buffer overnight at 4°C while gently agitating.
- Wash 3 times in TBST while gently agitating for 10 minutes per wash.
- Incubate the membrane with secondary antibody in blocking buffer for 1-2 hours at room temperature (RT) while gently agitating.
- Wash 3 times in TBST while gently agitating for 10 minutes per wash.
- Incubate the membrane at RT for 1 minute in mixture (1:1) of two ECL solutions.
- Remove the excess liquid and wrap the membrane in transparent plastic wrap.
- Expose to film and develop the image.