Introduction to Antibodies, 2 nd Edition. Stop the reaction with PBS containing 20 mM EDTA. Develop the blot at room temperature until the bands have darkened (approx. Add 10 ml of solution per 15X15 cm 2 membrane. For example, with an alkaline phosphatase labeled secondary antibody, add 66 µl of NBT stock to 10 ml of alkaline phosphatase buffer with mixing, then add 33 µl of BCIP stock.

Immediately prior to use, prepare fresh reagent for visualization and develop the blot.

Drain the reagent and blot the membrane on a clean piece of paper towel.

Wash the membrane for 5 minutes with PBS.

Incubate the membrane in the secondary antibody solution for 1 hour at room temperature. The concentration should be between 0.5 to 5 µg/ml, or a dilution of 1/200 to 1/2000 of a 1 mg/ml stock.

Prepare a dilution of the secondary antibody in blocking solution.

add 10 µl of a 1 mg/ml antibody to 10 ml of blocking solution for a 1 µg/ml dilution.) Incubate for a minimum of 1 hour at room temperature or overnight at 4☌ with gentle agitation.

Dilute the primary antibody in blocking solution to a concentration between 1 µg/ml and 50 µg/ml.

Rinse the membrane with PBS twice for 5 minutes.Incubate the membrane in blocking solution for 30 minutes to 2 hours at room temperature with gentle agitation.Wash the membrane twice for 5 minutes with deionized water to remove gel and transfer buffer components and weakly bound proteins.A corner may be snipped off in order to retain the orientation of the membrane. Remove and disassemble the sandwich, removing the nitrocellulose membrane. Run the transfer for 45 minutes to 1.5 hours with a current of 0.8 mA/cm 2. Verify the orientation of the gel and membrane to ensure that the proteins migrate in the correct direction the membrane should be above the gel, facing the positive electrode. Sandwich the gel, blotting membrane and absorbent papers as follows: bottom plate (cathode) - 3 sheets of absorbent paper – gel – membrane – 3 sheets of absorbent paper – top plate (anode).

Remove any air bubbles by rolling a pipette across the membrane. Apply the membrane to the surface of the gel.Soak the gel, nitrocellulose membrane, and absorbent papers in transfer buffer for 5 minutes.Pre-wet the nitrocellulose membrane in deionized water.Cut a sheet of nitrocellulose paper and 6 sheets of absorbent filter paper to the exact size of the SDS-PAGE gel.Run an SDS-PAGE to separate the proteins on a gel.Appropriate primary and conjugated secondary antibodies.Blocking solution: 3-5% BSA in PBS with a small amount of Tween®20.Washing buffer: PBS with 0.1% Tween ®20.Transfer buffer: 0.04% SDS, 20% Methanol, 48 mM Tris, 39 M glycine.Finally, the protein is visualized by the addition of a reagent which reacts in the presence of the label on the secondary antibody. Next, the membrane is incubated with a primary antibody which binds to the target protein on the blot, followed by a conjugated secondary antibody which binds to the primary antibody. After the transfer step, the membrane undergoes a blocking step which covers the entire surface of the membrane with detergent solution to prevent non-specific binding of antibody to the membrane. The Western Blot technique is composed of 4 steps: 1) separation of proteins by gel electrophoresis, 2) transfer of proteins to a solid support, 3) detection of target protein, and 4) visualization of target protein. This immunoblotting method detects the presence and relative quantity of proteins, their molecular weight, and the efficiency of protein extraction. Our featured application notes below provide further details.Western blotting provides a semi-quantitative method to measure protein levels. Utilizing this substrate-free assay, researchers can achieve equivalent sensitivity to traditional chemiluminescence assays while consolidating western blot, ELISA, and other applications onto a single reader. Each type of detection has advantages and disadvantages that need to be considered when selecting a method.Īnother method that we have introduced the ScanLater™ Western Blot Detection System, enables first-of-its-kind western blot detection in a multi-mode microplate reader platform. For example chemiluminescence can be detected using x-ray film or digital imaging equipment while a fluorescent secondary antibody requires a fluorescence imager. These methods are described below and require different equipment for detection. There are several western blot methods to consider depending upon the secondary antibody used, detection of the target protein may be colorimetric, chemiluminescent, or fluorescent.

Steps of western blotting technique