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    Protocols
    Bioss is dedicated to helping you achieve exceptional results. Our top-notch scientific support team has worked hard to develop these protocols for all our applications. We hope these instructional aids assist you in your research!



    Western Blotting Protocolpdf iconDOWNLOAD A PDF

    This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

    Sample Preparation
    1. Lyse sample in the appropriate lysis buffer.
    2. Reduce and denature the sample by adding sample buffer and heat for 5 minutes at 95°C.
    3. Centrifuge at 16000 x g in a microcentrifuge for 5 minutes.
    Electrophoresis
    1. Load 20-30μg of lysate per lane (or 100ng of purified protein) along with a molecular weight marker.
    2. Prepare the running buffer and assemble the gel in the tank.
    3. Drain blocking buffer from slide.
    4. Run SDS-PAGE for 30 minutes at an initial voltage of 75V for 20 minutes, then 115V for 60 minutes.
    Transfer
    1. Prepare transfer buffer.
    2. 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.
    3. Assemble transfer stack.
    4. Run transfer, 100V for 70 minutes.
    5. Check the transfer with Ponceau or Coomassie stain.
    Immunoblotting
    1. Block the membrane by incubating for 1 hour in blocking buffer (TBST with 5% BSA or milk).
    2. Incubate the membrane with primary antibody in blocking buffer overnight at 4°C while gently agitating.
    3. Wash 3 times in TBST while gently agitating for 10 minutes per wash.
    4. Incubate the membrane with secondary antibody in blocking buffer for 1-2 hours at room temperature (RT) while gently agitating.
    5. Wash 3 times in TBST while gently agitating for 10 minutes per wash.
    Detection
    1. Incubate the membrane at RT for 1 minute in mixture (1:1) of two ECL solutions.
    2. Remove the excess liquid and wrap the membrane in transparent plastic wrap.
    3. Expose to film and develop the image.