Western blot protocol

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Western blot (WB) protocol

1. Sample Preparation:
   • Collect and prepare the samples of interest (cell lysates, tissue lysates, or purified proteins).
   • Determine the protein concentration using a suitable method (e.g., Bradford assay or BCA assay).
2. Protein Separation by SDS-PAGE:
   • Prepare a polyacrylamide gel with the desired percentage of acrylamide (typically 8-15% resolving gel).
   • Prepare a stacking gel with a lower percentage of acrylamide (e.g., 4%).
   • Load equal amounts of protein samples onto the gel, along with a protein molecular weight marker.
   • Run the gel at a constant voltage until the dye front reaches the bottom of the resolving gel.
3. Protein Transfer to a Membrane:
   • Prepare a transfer buffer (commonly Tris-glycine or Tris-glycine-methanol buffer) and cool it in a cold room or on ice.
   • Cut a suitable membrane (e.g., nitrocellulose or PVDF) according to the gel size.
   • Soak the membrane in transfer buffer for a few minutes.
   • Set up a transfer apparatus with the gel, membrane, and filter paper soaked in transfer buffer.
   • Place the gel and membrane sandwich into the transfer apparatus, ensuring there are no air bubbles.
   • Run the transfer at a constant voltage (typically 100 V) for 1-2 hours (or according to the manufacturer’s instructions).
4. Membrane Blocking:
   • Prepare a blocking solution such as 5% non-fat milk or BSA in a suitable buffer (e.g., TBS or PBS).
   • Incubate the membrane in the blocking solution for 1-2 hours at room temperature or overnight at 4°C on a shaker.
5. Primary Antibody Incubation:
   • Dilute the primary antibody specific to the target protein in a suitable dilution buffer (e.g., 1% BSA in TBS-T or PBS-T).
   • Incubate the membrane with the primary antibody for 1-2 hours at room temperature or overnight at 4°C on a shaker.
6. Washing:
   • Wash the membrane three times for 10 minutes each with a suitable washing buffer (e.g., TBS-T or PBS-T) to remove unbound primary antibody.
7. Secondary Antibody Incubation:
   • Dilute the appropriate secondary antibody (conjugated to an enzyme or fluorophore) in a suitable dilution buffer.
   • Incubate the membrane with the secondary antibody for 1 hour at room temperature on a shaker.
8. Washing:
   • Wash the membrane three times for 10 minutes each with a suitable washing buffer to remove unbound secondary antibody.
9. Detection:
   • For enzyme-conjugated secondary antibodies, develop the signal using a suitable detection method (e.g., chemiluminescence or chromogenic substrate).
   • For fluorescently-labeled secondary antibodies, visualize the signal using a suitable imaging system (e.g., fluorescence scanner or imager).
10. Data Analysis:
    • Quantify the signal intensity using appropriate software.
    • Analyze the band sizes relative to the molecular weight marker.
    • Compare the protein expression levels among different samples or experimental conditions.

Remember to always refer to the specific protocols and recommendations provided by the manufacturers of the reagents and equipment you are using, as they may have specific instructions or variations for their products.