The optimal dilution of the anti-species antibody should be determined by the user. However, the final dilutions below should yield acceptable results for the respective applications.
Fluorescent western blotting: 1:15000
In-Cell Western: 1:2000
NOTE: The TrueBlack® Fluorescent Western Blot Blocking Buffer Kit (#40683) contains the necessary buffers to block the membrane and dilute the primary and secondary antibodies.
NOTE: Two-color western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. Overlap of epitopes may cause interference and should be considered in two color western blots. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) is recommended to verify electrotransfer and to determine molecular weights. Prestained markers are autofluorescent at near-infrared wavelengths.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
Warm the TrueBlack® WB Blocking Buffer to room temperature and thoroughly mix prior to use.
NOTE: The TrueBlack® WB Blocking Buffer may have a slight precipitate but this will not impact the performance.
Drain membrane of excess TBST and allow to dry.
CRITICAL STEP: Membrane must be dry for fluorescent staining.
Protocol Id: 2544
This antibody is prepared from goat antibodies and purified by immunoaffinity chromatography using antigen coupled to agarose beads.
Near infrared anti-species IgG conjugates are ideal for fluorescent western blotting and In-Cell Western. Cell Signaling Technology's strict quality control procedures assure that each conjugate provides optimal specificity and fluorescence.
This product has been optimized for use as a secondary antibody in fluorescent western blotting and In-Cell Western™.
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