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