Western Blot - 4. Secondary Reporter Antibody Reagents

Secondary antibody conjugates are ideal for western blotting. When choosing a secondary antibody conjugate for an antibody assay consideration must be given to target species, conjugate (i.e. peroxidase, FITC, Biotin) and host species. In addition to standard secondary antibodies, Rockland offers pre-adsorbed secondary antibodies which are suitable for detection methods where cross-reactivity may be an issue.  

Enzymatic secondary antibody conjugates:

Reporter enzymes are used extensively in molecular biology because they allow visualization or detection of immune complexes. Horseradish Peroxidase (HRP) is a widely used reporter enzyme, and depending on the substrate it can yield a chromogenic, or luminescent product.  Alkaline phosphatase is also used, most typically as the reporter in chromogenic western blot assay format. 

Fluorescent secondary antibody conjugates:

Rockland conjugates a broad group of secondary antibodies to many of the classic and next generation of fluorescent markers including fluorescein, Texas Red, Phycoerythrin. Rockland also produces many next generation flurochrome dyes. These are designed for detection of primary antibodies in multiplex, multi-color analysis.  Next generation fluorochrome conjugates (Atto-tec dyes, DyLight dyes) offer superior absorption (high extinction coefficient), high fluorescence quantum yield and superior high photostability. All of the conjugates are ideal for various immunofluorescence based assays including fluorescent western blotting, immunofluorescence microscopy, FLISA, and more. 

Biotin-Streptavidin secondary reporter reagents

Streptavidin is a protein purified from the Streptomycesavidinii.  The streptavidin tetramers have very high affinity for biotin, and biotin-streptavidin is one of the strongest non-covalent interactions observed in nature. Streptavidin conjugated to enzymatic or flourogenic reporters are used extensively in molecular biology and bio nanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants, and detergents.  Signal amplification is also a benefit of using this reporter system which can improve upon sensitivity when compare to typical antibody-target interactions. 

Browse all our secondary reporter antibody reagents 

Western Blot Protocol

1. Background
2. Types of Western Blot Formats
3. Primary Antibody Reagents
4. Secondary Reporter Antibody Reagents
5. Substrates
6. Buffers and Blocking Reagents
7. Western Blot Preassembled Kits