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Highly sensitive, fluorescence-based quantitation of purified proteins.
AccuOrange™ Protein Quantitation Kit is a highly sensitive fluorescence-based assay for quantifying purified protein samples in 96-well format.
The detection range of the assay is 0.1-15 ug/mL protein. AccuOrange™ is much more sensitive than traditional protein quantitation assays such as BCA, Bradford and Lowry, and shows superior linearity and reproducibility compared to the NanoOrange® protein quantitation assay. The assay shows minimal variability between different proteins, and has stable fluorescence signal for up to 16 hours.
AccuOrange™ is recommended for quantifying purified protein or antibody samples. The tolerance of the AccuOrange™ assay to salts, buffers, detergents, and other chemicals is listed in the product protocol. The AccuOrange™ assay has low tolerance for non-ionic detergents, and is not recommended for use with cell lysates containing Triton™ X-100, sodium deoxycholate, CHAPs, or other non-ionic detergents. The assay can tolerate up to 0.01% SDS (final concentration in assay).
Even though AccuOrange™ buffer does contain SDS, which is required for the dye to bind proteins, the assay is very sensitive to small changes in SDS concentration, and also cannot tolerate non-ionic detergents that form mixed micelles with SDS, like Triton®. Therefore we don't recommend using the kit for cell lysates or other samples with significant amounts of detergents.
Gels stained with One-Step Blue® can be dried just like gels stained with Coomassie. The stain will not interfere with the detection of radiolabeled proteins.
The AccuOrange™ assay is a fluorescent dye-based assay. The dye binds to proteins primarily through hydrophobic interactions. Proteins denature upon heating; the dye binds to the exposed hydrophobic pockets of the protein after cooling. The free AccuOrange™ dye is fluorogenic due to non-radioactive decay but becomes highly fluorescent due to the rigid conformation inside the pocket.
The AccuOrange™ assay more sensitive than traditional protein quantitation assays such as BCA, Bradford and Lowry, and shows superior linearity and reproducibility than the NanoOrange® protein quantitation assay (Thermo Fisher Sci.), but has low tolerance for detergents like SDS and Triton® X-100.