Protein Detection & Analysis
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.
Use of pre-stained protein markers can quench fluorescent protein stains. Switching to an unstained marker should resolve the issue.
We do not recommend staining proteins on the polyacrylamide gel with the One-Step protein gel stains before western blotting as it can significantly reduce protein transfer to the membrane. If protein detection on the gel prior to transfer is desired, the Total Protein Prestains would be more suitable.
The 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. After electrophoresis, the bands can be visualized on the gel using a fluorescent gel scanner, allowing detection of as little as 1 ng protein per band. Labeled proteins also can be transferred to membranes for western blotting. 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 and staining is reversible.
Lumitein™ is a luminescent dye designed for detecting proteins in SDS polyacrylamide (SDS-PAGE) gels. It can also be used to detect proteins in native PAGE gels after an additional SDS incubation step as described in the protocol PI-21002.