Secondary Antibody Protocols and Resources

Overview

Below you will find links to a selection of protocols and useful resources for various applications using secondary antibodies.

For our full set of resources please go to our antibody resources page.

Immunohistochemistry

• Protocol – general immunostaining protocol, with links to other protocols
• Tips for when using a secondary antibody in IHC

Mouse spleen cryosection stained with Rat Anti-Mouse F4/80 Antibody, clone Cl:A3-1 (MCA497G) at a 1/100 dilution followed by Goat Anti-Rat IgG:HRP (STAR72) at a 1/50 dilution.

Western Blotting Quick Tips

• How to Choose Secondary Antibodies for Multiplex Western Blotting

Western Blotting

• Protocol – general western blotting protocols
• Fluorescent antibodies – StarBright™ Blue 700 Antibodies (targeting mouse and rabbit) are ideal for the detection of single target proteins and multiplex detection of several proteins
• Detection of phosphorylated proteins protocol – a protocol that provides guidelines and compatible reagents when using Phospho-Specific PrecisionAb Antibodies
• Detect your protein of interest without interference from IgG chains – protocol for TidyBlot Western Blotting Detection Reagent

Western blot analysis of HEK293 embryonic kidney cell line whole cell lysate probed with Mouse Anti-Human H-Ras Antibody (MCA2884) followed by HRP conjugated Goat Anti-Mouse IgG Secondary Antibody (STAR117P) visualized using chemiluminescence.

ELISA

• Protocol – general indirect ELISA protocol, with links to competitive ELISA protocol
• Tips for ELISA, including antibody considerations

Sandwich ELISA analysis of human IgE using Mouse Anti-Human IgE Antibody (0100-0413) as a capture reagent and biotinylated Goat Anti-Human IgE Antibody (STAR147B) as a detection reagent, with recombinant human IgE (HCA171) as antigen for the generation of the standard curve. Detection is by HRP-conjugated streptavidin and substrate. The microtitre plate is read at O.D. 450 nm on the Bio-Rad iMark™ Microplate Absorbance Reader (1681135). Plasma and serum samples diluted 1:5 are indicated in green and orange, respectively.

Flow Cytometry

• Guidelines for the use of secondary detection reagents in flow cytometry to ensure specific and optimized staining
• Protocol – general indirect immunofluorescence staining of cells and blood protocol
• Products, protocols, tips and tricks for all your flow cytometry needs

A, RPE conjugated Rat Anti-Dog CD4 (MCA1038PE) and Mouse IgG1 Isotype Control (MCA928) labeled with Goat Anti-Mouse FITC (STAR117F). B, RPE conjugated Rat Anti-Dog CD4 (MCA1038PE) and Mouse Anti-Dog CD3 (MCA1774GA) labeled with Goat Anti-Mouse FITC (STAR117F). All experiments performed on red cell lysed canine blood gated lymphoid cells in the presence of 10% dog serum. Data acquired on the ZE5™ Cell Analyzer.

Browse application specific web pages to quickly and easily find antibodies, kits and reagents tested in your application of choice, and access many scientific tools such as protocols, webinars, tips and in-depth guides, all in one place.