Biochemical Techniques and Instrumentation

5.3.2.5.1. Methods of Detection

5.2.3.5.1. Methods of Detection

1. Enzyme linked secondary antibody

Two enzymes are commonly used for the detection. They are

• Alkaline phosphatase, which converts colourless 5 bromo 4 chloro indolyl phosphate (BCIP) subtrate into a blue product.
• Horse radish peroxidase (HRP), which with hydrogen peroxide as a substrate, oxidises either 3 amino 9 ethyl carbazole into an insoluble brown product or 4 chloro-1-napthol into an insoluble blue product.

2. Chemiluminescence (ECL) detection

In the presence of hydrogen peroxide and chemiluminescent substrate luminol, HRP oxidizes the luminol with concomitant production of light. The light intensity can be increased 1000 fold with a chemical enhancer. The light emission is then detected by exposing the blot to a photographic film. ECL substrates are also available for use with alkaline phosphatase labelled antibodies.

3. Radio labelled detection

1. ¹²⁵ I-labelled secondary antibody binding to the blot is detected by autoradiography
2. Fluorescine-labelled secondary antibody binding to the blot is detected for the fluorescent label by exposing them to the UV light
3. ¹²⁵I–labelled protein A. The protein A is purified from Staphylococcus aureus and specifically bound to the Fc region of IgG molecule. This protein binding to the blot is detected by autoradiography

4. Biotinylated secondary antibodies.

Biotin is a small molecular weight vitamin that binds strongly to the egg protein avidin. Blot is incubated with biotinylated secondary antibody and then incubated with enzyme conjugated avidin. Multiple biotin molecules can be linked to a single biotinylated antibody molecule and this provides an enhancement of the signal. The enzyme used is usually alkaline phosphatase or horse radish peroxidase.

5. Quantum dots

These are engineered semiconductor nanoparticles with diameters of 2-10 nm, which fluoresce when exposed to UV light. Quantum dot nanocrystals comprise a semiconductor core of CdSe (Cadmium selenium) surrounded by a shell of ZnS (Zinc sulpide). This crystal is coated with an organic molecular layer that provides water solubility and conjugation site for biomolecules. Therefore, secondary antibodies will be bound to a quantum dot and the position of binding of the second antibody on the blot is identified by exposing the blot to UV light.

6. Probes

Probes are also used to identify proteins. Radioactively labelled DNA probes can be used to detect DNA binding proteins on a blot. Blot is first incubated in a solution of radio labelled DNA, then washed to make an autoradiograph of the blot. The presence of radioactive bands is then detected on the autoradiograph and used to identify the positions of the DNA binding proteins on the blot.