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.
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 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 (or longer for increased sensitivity) with secondary gold conjugate diluted 1:10-1:200 times with membrane blocking solution for preparation.
- 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 streptavidin gold conjugate (top left) and streptavidin silver conjugate (top right) before and after enhancement using silver enhancement kit for membranes. Bottom picture illustrates and highlights the difference in appearance (color) of 50nm anti-human IgG noble metal nanoparticle conjugates prepared using NHS-activated gold nanoparticles, NHS-activated gold nanourchins and NHS-activated silver nanoparticles, respectively.
References
1. M. Moeremans, et al., Journal of Immunological Methods, 1984, 74, 353