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Chemiluminescence Detection Reagent for Western Blotting
The Chemiluminescence HRP Substrate Solution Kit (Product No. C4087) is a chemiluminescent detection reagent for horseradish peroxidase (HRP)-labeled probes bound to proteins on membranes. The two-component type is used by mixing 1:1 immediately before use.
Product
Example of Use
- Allow C4087 to reach room temperature.
- Add HRP-conjugated antibody to the blotted membrane and wash.
- Mix equal volumes of Solution A and Solution B.
* Approximately 0.1 mL of mixture per 1 cm2 of membrane. - Blot off excess wash buffer from the membrane.
- Place the membrane on plastic wrap or a clear polyethylene sheet.
- Pour the mixture over the membrane.
- Allow to react for 60 seconds at room temperature.
- Remove excess mixture.
- Detect chemiluminescence.
* Adjust exposure time according to signal intensity.
Figure 1. Membrane luminesced with C4087
HeLa Cell Lysate: 2.5 - 0.039 μg/lane (2x step dilution)
Membrane: PVDF membrane
Blocking Buffer: 1% BSA/TBS-T
Primary Antibody: Anti-GAPDH (Mouse IgG)
Secondary Antibody: Goat anti-Mouse IgG HRP
Detection: CCD Imager
Exposure Time: 120 seconds
Antibody Stripping Solution for Western Blotting
Western Blot Stripping Buffer (Product No. W0024) is used to strip antibodies from membranes that have undergone chemiluminescence detection. The antigen is retained on the membrane because the procedure is performed under mild conditions. This allows chemiluminescence detection to be repeated with a different antibody.
Product
Example of Use
- Separate 2-fold step dilutions of HeLa cell lysate (5.0 - 0.078 μg/lane) by SDS-PAGE.
- After Western blotting, detect antibodies using chemiluminescence reagents. (Figure 2)
- Immerse the membrane in TBS-T and shake for 10 minutes. Repeat this procedure twice.
- Immerse the membrane in W0024 and shake for 30 minutes at room temperature. (Figure 3)
- Immerse the membrane in TBS-T and shake for 10 minutes. Repeat three times.
- Start the blocking procedure again and detect the new antibody by chemiluminescence. (Figure 4)
Figure 2. First detection. Detection by binding of Anti-αTubulin Antibody (Rabbit IgG)
Blocking Buffer: PVDF membrane
Blocking Buffer: 1% BSA/TBS-T
Wash Buffer: TBS-T
Primary Antibody: Anti-αTubulin (Rabbit IgG)
Secondary Antibody: Goat anti-Rabbit IgG HRP
Detection: CCD Imager
Exposure Time: 60 seconds
Figure 3. Antibody removal with W0024
Figure 4. Second detection. Detection by re-blocking the stripped membrane and binding Anti-GAPDH Antibody (Mouse IgG)
Blocking Buffer: 1% BSA/TBS-T
Wash Buffer: TBS-T
Primary Antibody: Anti-GAPDH (Mouse IgG)
Secondary Antibody: Goat anti-Mouse IgG HRP
Detection: CCD Imager
Exposure Time: 60 seconds