Merck

Immunodetection Reagents & Substrates

Immobilon® Forte, Crescendo, and Classico Chemiluminscent Western Blotting Substrates

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. 


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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.

Western Blot Blocking Reagents Selection Guide

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. We also offer a broad selection of HRP and AP substrates for chemiluminescent, chromogenic, and chemifluorescent detection in Western blotting.

CHEMILUMINESCENT SUBSTRATES FOR WESTERN BLOTTING

Immobilon® HRP substrates for chemiluminescent detection provide increased sensitivity for more quantitative blots, reducing costs by enabling you to decrease the concentration of antibody used for detection. Choose from a portfolio of Immobilon® substrates with varying sensitivities in convenient ready-to-use or two component formats to optimize detection for your Western blotting application.

  • 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 sensitivities to provide optimal signal-to-background ratios across a spectrum of target protein concentrations. Detection limits:
    • Immobilon® Classico: ~ 6 pg of target protein
    • Immobilon® Crescendo: ~ 1-3 pg target protein
    • Immobilon® Forte: ~ 400 fg target protein
       
  • Immobilon® Western HRP, Immobilon® ECL Ultra, and Immobilon® UltraPlus Western substrates deliver exceptional sensitivity and long signal life in standard 2-component formats. These formulations permit the use of more dilute primary antibody solutions for immunoblot detection. Detection limits:
    • Immobilon® Western HRP substrate: ~ 400 fg target protein
    • Immobilon® ECL Ultra substrate: low femtogram range
    • Immobilon® ECL UltraPlus Western Blot HRP Substrate: low femtogram range

Relative Sensitivity of Immobilon® Chemiluminescent HRP Substrates

Two-fold dilution showing the relative sensitivity of Immobilon® chemiluminescent substrates for Western blotting, including: Classico (lower sensitivity), Crescendo, Forte, Ultra, and UltraPlus (highest sensitivity).

Figure 1. Two-fold dilution series of A431 cell lysate [12-110] starting at 4 μg protein was run on 10% mPAGE® gels [MP10W15] in MOPS buffer [MPM0PS] at 200V for 25 minutes. Protein was transferred to Immobilon®-P membrane [IPVH00010] for 1 hour in mPAGE® transfer buffer [MPTRB] with 10% ethanol. Immunodetection of HH3 protein (15 kDa) was performed with rabbit anti-HH3 [H0164] and goat anti-rabbit [AP132P] antibodies at listed dilutions. Blots were incubated with indicated ECL reagents for 5 minutes and images were taken with a 30s exposure on Bio-Rad® ChemiDoc system.

Cytiva™ Amersham™ ECL detection reagents use enhanced luminol-based HRP detection, with different formulations available to cover different Western blotting application needs. 

  • Cytiva Amersham™ ECL Western blotting detection reagents are recommended for confirmatory Western blotting applications.
  • Cytiva Amersham™ ECL Prime Western blotting detection reagent enables detection of both high and low abundant proteins on the same blot.
  • Cytiva Amersham™ ECL Select Western blotting detection reagents offer high sensitivity even when using diluted primary and secondary antibodies.
  • Cytiva Amersham™ ECL Start Western Blotting detection reagent was developed for chemiluminescent detection of medium- and high-expression proteins.

CHROMOGENIC SUBSTRATES FOR WESTERN BLOTTING

Chromogenic detection utilizes a peroxidase (e.g., HRP) or alkaline phosphatase (AP) conjugated enzyme to catalyze a reaction that results in the deposition of an insoluble colored precipitate for detection. Our chromogenic substrates for Western blotting are available in solution, powder, and tablet form to provide flexible options for storage and usage.

  • SIGMAFAST™ DAB Tablets (chromogenic HRP substrate) 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 (chromogenic AP substrates) are highly sensitive colorimetric substrates for alkaline phosphatase.
  • TMB Enhanced One Component HRP Membrane Substrate (chromogenic HRP substrate) is a single component 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.

  • ReBlot™ Plus reagents efficiently strip probed blots of bound antibodies. ReBlot™ Plus reagents are available in mild and strong formulations.


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