Immunoblotting
Due to the relative ease of preparing antibody gold conjugates, and straightforward detection of gold nanoparticles by naked eye without prior development procedures, these probes has found great use in rapid tests such as lateral flow, western blot and dot-blot assays.
When membranes of blotted proteins are probed with a secondary gold conjugate, the presence of a target protein is indicated with a red color upon binding of the gold conjugate.
When combined with silver enhancement (i.e. deposition of silver onto bound gold colloid turning the spot dark in color) sensitivity in western blot and dot-blot applications rivals that of colorimetric detection methods. In addition, secondary gold probes adapt well to standard western blot protocols and little changes are necessary to your current detection scheme.
Immunogold Dot-Blot Related Products
- Our Gold Nanoparticles for Immunoblotting have an ideal size for use in immunoblotting assays, and conjugates can easily be prepared by simple adsorption of antibodies to the gold surface.
- Carboxyl Functionalized Gold Nanoparticles - Suitable for covalent attachment of antibodies to the gold surface.
- Secondary Antibodies and Conjugates
- Silver Enhancement Kit - Suitable for signal enhancement and down to pg detection of target.
- Gold Nanoparticle Conjugation Optimization Kits - Complete systems to optimize conjugation of proteins to gold nanoparticles.
Standard Immunogold Dot-Blot Protocol
(Adapted from Moeremans et al. [1])- Spot one microlitre drops of a serial dilution of your protein (100-0.1ng) in PBS supplemented with 50ug/ml of BSA on nitrocellulose or PVDF membrane.
- Let protein drops dry into the membrane.
- Block with Cytodiagnostics Membrane Blocking Solution for 30 minutes at room temperature.
- Inubate with primary antibody for 2 hours at room temperature.
- Wash membrane 3x5 minutes with membrane blocking solution.
- Incubate for 2 hours with secondary gold conjugate diluted 1:50-1:200 times with membrane blocking solution (see Tech Note #102) for preparation.
- Wash 3x5 minutes as above.
- Dry membrane and record data.
- (OPTIONAL) Proceed with silver enhancement to improve sensitivity
References
1. M. Moeremans, et al., Journal of Immunological Methods, 1984, 74, 353
