Immunoblotting Using Noble Metal Nanoparticle Conjugates
Due to the relative ease of preparing antibody conjugates of noble metal nanoparticles such as gold and silver and the straightforward detection by naked eye without prior development procedures, these probes have found great use in many assays. Applications include rapid tests such as lateral flow, vertical flow, western blots and dot-blot assays. In addition, the unique optical properties of each noble metal nanoparticle type allows for generating secondary probes with different colours that can be utilized for colorimetric multiplexing, figure 1.
When membranes of blotted proteins are probed with a noble metal protein conjugate such as a secondary gold conjugate, the presence of a target protein is indicated with a red colour upon binding of the nanoparticle probe, figure 1. When combined with silver enhancement (i.e. deposition of silver onto the bound gold conjugate) sensitivity in western blot and dot-blot applications rivals that of colorimetric detection methods. In addition, secondary noble metal nanoparticle protein conjugates adapt well to standard western blot protocols and little changes are necessary to your current detection scheme.
Immunoblotting with noble metal nanoparticle protein conjugates have several advantages over traditional detection probes such as:
- No development required for detection
- No expensive imaging equipment required for detection
- Allows for colorimetric multiplexing
Below is a standard dot-blot protocol for detection of an antigen on a membrane using a secondary gold conjugate. The same protocol can be used with gold nanourchins, silver nanoparticles, and alloy nanoparticle protein conjugate probes.
Standard Immunogold Dot-Blot Protocol
(Adapted from Moeremans et al. )
- Spot one microliter drops of a serial dilution of your protein (0.1 - 100 ng) in PBS supplemented with 50 ug/ml of BSA on nitrocellulose or PVDF membrane.
- Let protein drops dry into the membrane.
- Block Membrane for 30 minutes using 1% (w/v) dry milk in 1X PBS at room temperature.
- Incubate with primary antibody for 2 hours at room temperature.
- Wash membrane 3x5 minutes with blocking solution prepared as above.
- Incubate for 2 hours (or longer for increased sensitivity) with secondary gold conjugate diluted 1:10 (OD=0.3) with 0.2% dry milk. Note: See Tech Note #102 for preparation of a gold conjugate.
- Wash 3x5 minutes as above.
- Dry membrane and record data.
- (OPTIONAL) Proceed with silver enhancement to improve sensitivity.
Figure 1. Example dot-blot assay for Cytodiagnostics streptavidin gold conjugate (top left) and our streptavidin silver conjugate (top right) before and after enhancement using Cytodiagnostics silver enhancement kit for membranes. Bottom picture illustrates simultaneous multiplex detection of three different antigens using a mixture of noble metal nanoparticle conjugates with different optical properties, i.e. anti-human IgG 30nm gold/silver (20/80) alloy conjugate (green), a-mouse IgG 30nm gold/silver (80/20) alloy conjugate (red) and a a-rabbit IgG 40nm gold conjugate (purple).
Figure 2. Multiplex detection in a vertical flow dot blot immunoassay. Left image shows positive detection of 2 out of 3 antigens (red dots) and the control. The right image shows positive detection of two different antigens using two nanoparticle probes with different colours (red: gold nanoparticles, blue: gold nanourchins.
Figure 3. Western blot detection of purified actin using a rabbit anti-actin primary antibody followed by secondary detection using a 10nm anti-rabbit IgG gold conjugate and enhancement using Cytodiagnostics silver enhancement kit for membranes.
Immunoblotting Related Products
1. M. Moeremans, et al., Journal of Immunological Methods, 1984, 74, 353