WESTERN BLOTTING

Near-infrared (near-IR) western detection is highly sensitive, and offers advantages of wider linear range and multiplexing capability compared to chemiluminescence detection (see our webinar to learn more). Biotium’s near-IR CF® dyes are the brightest and most photostable available. Learn more about CF®680 and CF®770 dyes for near-IR western, In-Cell Western®, and other applications.

The TrueBlack® WB Blocking Buffer Kit is a ready-to-use buffer system for fluorescence-based western blotting (WB). The buffers yield optimal specificity and sensitivity by blocking non-specific interactions of dye-labeled antibodies with proteins and the blotting membrane.

TrueBlack® WB Blocking Buffer Kit Features

• Blocks as well or better than Odyssey® Blocking Buffer, at a lower price
• Reduces non-specific protein bands and background over entire membrane
• Suppresses background from charged dyes better than BSA, gelatin, or casein
• Compatible with PVDF and nitrocellulose membranes
• Contains no mammalian proteins, for broad antibody compatibility
• For visible and near-IR fluorescent westerns

Superior western blocking for next-generation fluorescent dyes

Non-specific signal in WB can arise from multiple sources, including antibody cross-reactivity with off-target proteins, non-specific antibody adsorption to the membrane, and membrane autofluorescence. Another potential cause of background is the effect of fluorescent dyes themselves on the specificity of labeled antibodies. Highly charged dyes like Alexa Fluor® or CF® dyes have improved solubility and brightness of conjugates compared to uncharged dyes. However, the extra charge carried by antibodies labeled with these dyes can result in non-specific binding to proteins and membranes. The TrueBlack® WB Blocking Buffer Kit blocks background from multiple sources including charged dye conjugates (Figure 2). TrueBlack® blocking buffer is especially advantageous for phosphoprotein detection, significantly improving specificity compared to conventional blocking buffers (Figure 1).

Switch from Odyssey® Blocking Buffer and save

TrueBlack® WB Blocking Buffer performs as well or better for fluorescent WB compared to LI-COR’s Odyssey® Blocking Buffer (Figure 1), and is priced lower on a per membrane basis.

Compare TrueBlack® WB Blocking Buffer Kit with Odyssey® Blocking Buffer

VersaBlot™ Total Protein Normalization Kits allow simple, sensitive and highly linear protein quantitation on SDS-PAGE gels and western blot membranes. The kits allow you to label purified proteins or cell lysates with our near-infrared CF® dyes before running the samples on SDS-PAGE. Proteins can then be visualized on a gel or membrane using a fluorescent gel scanner, allowing detection of as little as 1 ng protein per band. If desired, the prestain can be reversed after scanning, facilitating downstream multi-color western blot analysis. The staining demonstrates excellent linearity for quantitation of total protein over a wide dynamic range, outperforming traditional western blot normalization based on housekeeping protein detection.

RedDot™1 is a far-red cell membrane-permeable nuclear dye similar to Draq5™. Staining does not require a wash step and demonstrates greater photostability than the traditional blue fluorescent nuclear stains DAPI and Hoechst. RedDot™1 can be excited at a wide range of wavelengths between 488 nm and 647 nm, with an emission maximum at 694 nm. RedDot™1 is particularly useful for cell number normalization for In-Cell Western® assays using a near-infrared scanner such as the LI-COR® Odyssey®. In fixed cells, RedDot™ 1 staining generates a highly linear fluorescence signal in the 700 nm channel that is proportional to the number of cells. In a direct comparison with DRAQ5™/Sapphire700™,  RedDot™ 1 staining demonstrates a higher signal and a wider linear range.