Related Experiment Video
Updated: Feb 6, 2026

Western Blotting: Sample Preparation to Detection
Published on: October 14, 2010
Development of a western blot method for detection of fish ectoparasite Argulus siamensis antigens
P Das1,2, J Mohanty1, M R Badhe1
1a Fish Genetics and Biotechnology Division , ICAR-Central Institute of Freshwater Aquaculture , Bhubaneswar , India.
Abstract:
A study was undertaken to develop a western blot method for detection of immunogenic proteins of fish ectoparasite, Argulus siamensis for its further use as potential vaccine candidates. Argulus antigens were prepared by homogenization and injected to rohu (Labeo rohita) juveniles for development of immune serum. The serum was used to immunostain the antigens in western blot. The other reagents added in sequence were rabbit anti-rohu serum, goat anti-rabbit ALP conjugate and substrate (BCIP-NBT). However, similar banding patterns were observed with both control and immunized rohu serum. Hence, a possible cross-reaction was suspected and verified in number of western blot experiments. A typical cross-reaction observed was of rabbit serum reacting directly with Argulus antigens. Hence, the rabbit anti-rohu serum was replaced with guinea pig anti-rohu serum. Another cross-reaction of goat anti-guinea pig ALP conjugate with rohu serum was eliminated by using goat anti-rabbit ALP conjugate with guinea pig serum. Thus, the final western blot method consisting of Argulus antigens → rohu serum → guinea pig anti-rohu serum → goat anti-rabbit ALP conjugate → substrate, yielded distinguishing results between control and Argulus-immunized rohu serum samples. The developed test has tremendous downstream applications, particularly in immunoproteomic studies of Argulus antigens.
Related Concept Videos
Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
Osmoregulation in Fishes
Development of Analytical Methods
Southern Blot
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Effects of EDTA on End-Point Detection Methods
In the visual method, metal-ion indicators (metallochromic dyes), which have distinct colors in their free and complex forms, are added to the mixture to signal the titration's end point. They form stable complexes with metal ions, but these complexes are weaker than the corresponding metal–EDTA complexes. As a...
Precipitation Titration: Endpoint Detection Methods
In the Volhard method, a standard excess of AgNO3 is first added to the...

