The first step in immunoblotting is to rinse and block the membrane with non-specific protein, such as milk or BSA. The purpose of this blocking step is to bind non-specific protein to the surface of the membrane where sample protein is not already present. This prevents antibody from binding non-specifically to the membrane, which would result in a high background signal. The choice of milk vs. BSA is antibody specific and may require optimization. Often the antibody information sheet will recommend one over the other.
Primary antibody: After blocking, the membrane is incubated in a solution containing the primary antibody. The primary antibody recognizes and binds the epitope or the specific amino-acid sequence of the protein of interest.
Secondary antibody: After washing to remove unbound primary antibody, secondary antibody is added. Secondary antibody recognizes the primary antibody. Secondary antibodies used for western blotting are typically conjugated with an enzyme; the most commonly used enzymes are Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (AP).
Note: Some primary antibodies are directly conjugated to HRP, eliminating the need for the secondary antibody incubation steps. In this case, it is possible to proceed to detection after the primary antibody incubation and subsequent rinses. If elimination of the secondary antibody step is desired, Novus offers HRP conjugated primary antibodies and Lightning-Link Antibody Labeling Kits, which can be used to conjugate an unlabeled primary antibody to HRP or other desired conjugates.
After transfer, carefully disassemble the transfer stack and rinse the membrane briefly in distilled water or 1X TBST.
20 mM Tris base
150 mM NaCl
0.1% Tween 20
Gently mark MW ladder bands with a pencil for size detection. If all blue molecular weight markers were used, this step can be omitted as the bands of all blue markers will be visible after detection when used in conjugation with the Blue Marker Antibody.
If desired, stain the membrane with Ponceau red (a reversible protein stain) for 30 seconds to visualize protein bands to confirm that protein transfer was successful. Rinsing the membrane briefly with distilled water after Ponceau staining will reveal protein bands. Wash away Ponceau red with several washes in 1x TBST until membrane is clear. Additionally, Coomassie staining of the gel after transfer can help assure that proteins were completely transferred from the gel to the membrane (minimal or no protein staining should be visible on a coomassie-stained gel after successful complete transfer).
Incubate the membrane in blocking solution for 1 hour at room temperature or overnight at 4˚C with constant rocking.
5% non-fat dry milk or 5% BSA
Optional step: Rinse the membrane for 5 minutes in 1X TBST to further reduce background.
Dilute the primary antibody to working concentration in 1X TBST with 1% milk or BSA (depending on what was chosen for blocking).
Note: Typical working antibody dilutions range from 1:500 to 1:5000 or greater. For recommended dilutions, please see the antibody datasheet. However, optimal dilutions may need to be determined experimentally.
Optional step: To visualize the molecular weight markers in addition to the protein of interest, 1 ug/mL Blue Marker Antibody can be added into the primary antibody solution. This antibody does not cross react with protein lysates and will bind specifically to the blue dye of each molecular weight marker.
Incubate the membrane in primary antibody solution for 1-2 hours at room temperature or overnight at 4˚C with gentle rocking. The incubation time may require optimization. However, overnight incubation at 4˚C is generally recommended to reduce non-specific background signal.
Wash the membrane with 1X TBST three times for 10 minutes each with gentle rocking.
Tip: Increase the number of washes to 5-6 if high background occurs. To reduce non-specific background, please see our Troubleshooting Guide.
Incubate the membrane in the diluted secondary antibody (in 1X TBST and may include 1% milk or BSA) for 1 hour at room temperature with gentle rocking.
Note: See primary antibody information sheet for proper secondary antibody selection; the secondary antibody must recognize the host species of the primary antibody. Secondary antibody concentration guidelines are listed in the product datasheet. For more information on selecting the best secondary antibody for your experiment, please read our Secondary Antibody Handbook.
Wash the membrane in 1X TBST three times for 10 minutes each with gentle rocking.
TIP: Do not let the membrane dry at any point during the blotting process.
In Western blotting, the method of detection is dependent on which enzyme is conjugated to the secondary antibody (or in some cases the primary antibody). The most commonly used enzyme in western blotting is HRP. Once substrate has been added, it reacts with HRP and emits light. The emitted light is detected using autoradiography film or chemiluminescence imaging system.
Prepare the ECL substrate just prior to use according to the manufacturer’s instructions. Novus offers two ECL substrates: PicoTect and NovaLume. Due to the higher sensitivity, we recommend NovaLume to detect the expression of low abundance proteins.
Incubate the membrane in the substrate according to manufacturer’s directions. Typical incubation times are 1-5 minutes.
Tip: More sensitive substrates require shorter incubation times to achieve optimal signal and avoid overexposure.
Carefully remove the membrane from the detection reagent and sandwich it between layers of plastic (e.g. a sheet protector or plastic wrap).
Expose the membrane to autoradiography film in a dark room or image with a chemiluminescent imaging system, such as a ChemiDoc.
Tip: Clip the top right corner of your film as a guide for film orientation in a dark room.
Tip: Use multiple exposure lengths to identify the optimal exposure time. An initial 10 second exposure will indicate the necessary exposure time
The developed film or image can be lined up in the correct orientation over the blot in order to mark the MW ladder positions if the Blue Marker Antibody is not used.