Western blot protocol

      Fig. The schematic diagram of western bolt protocol.

      Western blot protocol procedure

      Sample prepare

      Usually, samples are resuspended or mixed in 20-50 µl of 1X sample buffer and all the mixtures are incubated at 90°C for five minutes or 50°C for 15 minutes to break down the interactions between peptides.

      Protein Concentration Determination

      The Bradford assay method (Bio-Rad Protein Assay) is utilized to determine the concentration of proteins in each sample. The reading obtained is compared to a standard curve that allowed for a relative measurement of protein concentration. The standard curve is produced by using varying concentrations of bovine serum albumin (BSA).

      SDS-PAGE

      SDS-PAGE is performed using the Mini-PROTEAN III electrophoresis system (Bio-Rad).

      • Samples are loaded and fractionated on 8, 10, or 12% polyacrylamide gels along with a protein ladder.
      • Gels are electrophoresed between 100 and 150 V in running buffer (3% Tris-HCl, 14.4% glycine, 1% SDS) until the bromophenol blue dye reaches the bottom of the gel.
      • Gels are either electrotransferred to PVDF membranes.

      Western Blot Analysis

      • The Mini Trans-Blot Electrophoretic Transfer Cell system (Bio-Rad) is used for western blot experiments.
      • Proteins fractionated by SDS-PAGE are electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The transfer is carried out in transfer buffer (192 mM glycine, 20% v/v methanol, 25 mM Tris-HCl, pH 8.3). The protein transfer is carried out at 100 V for 1 hour at 4°C with buffer recirculation. All of the remaining steps are carried out at room temperature.
      • After transfer, the membranes are blocked with a BSA blocking solution (5% BSA, 150 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5) or a milk blocking solution (5% powdered milk, 150 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5).
      • The membranes are incubated in appropriate dilutions of primary antibody in TBST (150 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5) for 1 hour at room temperature or overnight at 4°C, with carefully shaking. The primary antibody is used following the manufacturer’s advice.
      • The membranes are washed 3 times with TBST for 10 minutes remove free primary antibody.
      • For colorimetric detection, the secondary antibody alkaline, phosphataseconjugated α-rabbit IgG, is incubated with the membrane at appropriate dilution in TBST for 1 hour at room temperature.
      • The membrane is washed with TBST as before to remove any free secondary antibody.
      • An additional 30 second wash with TBS is carried out.
      • The antibodies are visualized by adding the color development substrates, Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP), in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris-HCl, pH 9.5). The reactions are terminated by washing the membranes in water.

      Chemiluminescence detection

      • For chemiluminescence detection, the secondary antibody, horseradish peroxidase-conjugated α-mouse IgG, is incubated with the membrane at a 1:10,000 dilution in TBST for 1 hour.
      • The membrane is washed with TBST as before to remove any free secondary antibody.
      • An additional 30 second wash with TBS is carried out.
      • The antibodies are visualized by mixing the chemiluminescence development substrates, Peroxide Buffer and Luminol/Enhancer Solution (Pierce), in a 1:1 ratio and incubating this mixture on the membrane for 5 minutes.
      • The membrane is immediately scanned using an imager following manufacturer’s instructions.