Western blotting secondary antibodies
Western blotting is a widely used technique for detecting and quantifying specific proteins in various samples, such as cell and tissue samples.
Secondary antibodies conjugated to enzymes or fluorophores enable indirect labeling visualization in the western blot assay. Detection is achieved via chemiluminescence or fluorescence, producing bands that indicate protein presence and quantity. This method is highly specific and sensitive, making it ideal for protein analysis. Indirect detection provides signal amplification, which is useful for studying low-expressed proteins.
Choosing conjugated secondary antibodies
To visualize your protein, select a secondary antibody (against the host species of the primary antibody) that will bind to the primary antibody. Using an enzyme-linked secondary antibody, such as horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-conjugate antibody, or a western blot-optimized fluorescence conjugate, offers a high level of sensitivity.
The increased sensitivity of conjugated secondary antibodies, compared to primary antibody only, results from these antibodies binding to the primary antibody at multiple locations, which amplifies the signal. Therefore, using secondary antibodies is ideal for western blot since their signal amplification allows for easier detection of the protein of interest in a complex protein mixture.
Fluorometric western blotting
Chemiluminescent western blotting
We offer a wide range of HRP-conjugated, Alkaline phosphatase (AP)- fluorescent dye conjugated, and unconjugated secondary antibodies to visualize the presence and quantity of your specific protein target.
Specificity and cross-reactivity
Our secondary antibodies are tested to provide high specificity and minimal cross-reactivity for optimal western blot analysis.
Broad compatibility
Our secondary antibodies are compatible with a range of primary antibodies from different species. This versatility ensures that researchers can integrate our products seamlessly into their existing western blot workflow.
Flexibility in detection
Apart from HRP-conjugation, we offer secondary antibodies conjugated with other enzymes and dyes to suit different detection systems and preferences, allowing you to choose the best option for your specific needs.
FAQ
What makes HRP-conjugated secondary antibodies ideal for western blotting?
HRP-conjugated secondary antibodies provide a high level of signal amplification through chemiluminescence, making them ideal for detecting low-abundance proteins and ensuring high sensitivity in western blotting.
Can I use Abcam’s secondary antibodies with primary antibodies from another supplier?
Yes, Abcam's secondary antibodies are designed to be compatible with primary antibodies from various sources, as long as the primary antibody is targeted against the appropriate species.
How should I store my HRP-conjugated secondary antibodies?
For optimal performance please follow the recommended storage instructions. Typically store HRP-conjugated secondary antibodies are stored at 4°C in the dark. Avoid repeated freeze-thaw cycles which may affect the antibody activity.
Are there alternative conjugates available for western blotting secondary antibodies?
Yes, apart from HRP, we offer several secondary antibodies conjugated with other enzymes and fluorescent labels, permitting flexibility in detection based on the experimental setup and requirements.