Within molecular and cellular biology, secondary antibodies have become essential tools, highlighting the complexities of immunodetection. These versatile proteins play a crucial role in amplifying signals and enhancing the sensitivity of various laboratory techniques, resulting in a deeper understanding of fundamental biological components. Researchers utilise secondary antibodies to delve into cellular structures and investigate the details of biomolecular interactions.
There is a plethora of different applications in which secondary antibodies can be utilised to benefit researchers in the immunodetection and immunological techniques for research.
Immunofluorescence (IF): Secondary antibodies labelled with fluorescent dyes are used to visualise the presence and localisation of specific antigens within cells or tissues. By binding to the primary antibodies targeting the antigen of interest, the secondary antibodies enable the visualisation of cellular structures and protein distributions under a fluorescence microscope.
Western Blotting: In a Western blot analysis, secondary antibodies are conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase. These are utilised to detect specific proteins separated by gel electrophoresis. The secondary antibodies bind to primary antibodies that have attached to the target protein bands, allowing the visualisation of protein bands via enzymatic reactions.
Immunohistochemistry (IHC): Secondary antibodies labelled with enzymes or fluorophores are used to identify and localise specific antigens within tissue sections. This technique is often used in medical research and diagnostics to visualise protein expression and distribution within tissues.
Enzyme-Linked Immunosorbent Assay (ELISA): Secondary antibodies conjugated to enzymes are used in ELISA to detect the presence and quantity of antigens or antibodies. They facilitate the generation of a measurable signal, usually a colour change, which is proportional to the amount of target molecules present in the sample.
Flow Cytometry: Secondary antibodies conjugated to fluorescent markers are used in flow cytometry to analyse and quantify specific cell surface markers or intracellular proteins. This technique allows researchers to study individual cells within a heterogeneous population.
Multiplexing: Secondary antibodies with different fluorophores or enzymes can be used simultaneously in a single experiment to detect multiple target antigens. This allows researchers to study multiple proteins within the same sample, providing a comprehensive view of molecular interactions.
Immunoprecipitation: Secondary antibodies can be used to capture and isolate protein complexes by binding to primary antibodies attached to target proteins. This technique helps researchers study protein-protein interactions and identify associated molecules.
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