Immunodetection Reagents & Substrates

Western blotting reagents for detection

In Western blotting, the most important factor in determining the success of experiments is the quality of reagents used. We offer an array of Western blotting reagents that are pre-optimized to work synergistically, providing strong specific signals and low background to help you quickly produce publication-quality results.

Blocking Reagents

Blocking of unbound membrane sites prevents non-specific binding of antibodies that can lead to high background. Traditional milk, gelatin, and other protein-based blockers are effective for many blotting applications but can reduce sensitivity or detection by masking signal or interfering with detection of specific protein targets. Immobilon® Block Noise Cancelling Reagents include protein-free, ready-to-use buffers optimized to reduce background levels when using chemiluminescence, fluorescent, or phosphoprotein detection.

Streptavidin and Secondary Antibody Conjugates

Modern immunodetection methods are based on enzyme-linked detection using streptavidin or secondary antibodies covalently bound to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The conjugated enzyme catalyzes the degradation of specific substrates, resulting in signal generation. A variety of streptavidin, Protein A, Protein G, and secondary antibody conjugates are available for Western blotting.

Western Blotting Substrates

We offer a broad selection of substrates for enhanced chemiluminescent and colorimetric detection.

  • Immobilon® Classico, Crescendo and Forte Chemiluminescent substrates comprise a family of premixed, ready-to-use reagents for peroxidase-based detection offered in a range of sensitivies to provide optimal signal-to-background ratios across a spectrum of target protein concentrations. The detection limits are ~ 6 pg of target protein for Immobilon® Classico, ~ 1-3 pg for Immobilon® Crescendo, and ~ 400 fg for Immobilon® Forte.
  • Immobilon® ECL Ultra and Western Chemiluminescent HRP substrates
  • deliver exceptional sensitivity and long signal life in standard two-component formats. These formulations permit the use of more dilute primary antibody solutions for immunoblot detection. Immobilon® Western HRP substrate has a detection limit of ~ 400 fg. Immobilon® ECL Ultra substrate provides sensitivity at the low femtogram range with longer signal duration than other substrates in its class.
  • SIGMAFAST™ DAB Tablets are composed of a colorimetric precipitating substrate that produces a brown color upon reaction with horseradish peroxidase and can be dissolved in water to prepare a ready-to-use buffered solution for blotting detection.
  • BCIP/NBT (blue/purple) and BCIP/TNBT (blue/brown) Single Reagent are highly sensitive colorimetric substrates for alkaline phosphatase.
  • TMB Enhanced One Component HRP Membrane Substrate is a colorimetric substrate that produces a dark blue product.

Signal Enhancers

  • Immobilon® Signal Enhancer is designed for use in Western blotting applications when low signal intensity is observed. Enhancement of the antibody-antigen reaction can produce a 2 to 10-fold increase in signal intensity. Immobilon® Signal Enhancer is compatible with PVDF and nitrocellulose membranes and can be used with chromogenic, chemiluminescent, and fluorescent detection methods.
  • SignalBoost™ Immunoreaction Enhancer Kit is designed to enhance chemiluminescent or colorimetric target signal on nitrocellulose or PVDF membranes

Reagents and Kits for Stripping and Re-probing

Stripping and re-probing reagents are specially formulated to quickly and effectively remove antibodies from Western blots without significantly affecting the immobilized proteins.