Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate‐polyacrylamide gels

Abstract

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.

Analysis of Steady-state Protein Phosphorylation in Mitochondria Using a Novel Fluorescent Phosphosensor Dye

“…Western Blotting-Large format one-dimensional gels were electroblotted according to published protocols (27). Phosphoamino acid-specific antibodies were visualized by chemiluminescence using an ECL kit (Amersham Biosciencs).…”

Selective proteome‐wide detection of hydrophobic integral membrane proteins using a novel fluorescence‐based staining technology

“…2 and 4, SDS-polyacrylamide gels that have been stained with Pro-Q Amber dye to highlight proteins containing a-helical transmembrane domains may subsequently be post-stained with SYPRO Ruby protein gel stain in order to reveal the total protein profile. The feasibility of serially detecting three attributes in a single SDSpolyacrylamide gel was determined using Pro-Q Sapphire 532 oligohistidine tag stain, a newly developed stain for detecting oligohistidine-tagged fusion proteins that is closely related to previously described fluorescencebased stains [5]. This dye differs from Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine stains with respect to its spectral properties.…”

“…After staining gels in Pro-Q Amber stain, gels may next be stained with Pro-Q Sapphire 532 stain for visualization of oligohistidine-tagged fusion proteins, essentially as previously described [5]. Briefly, gels were placed directly into Pro-Q Sapphire 532 staining solution.…”

New building blocks or dendritic pseudopeptides for metal chelating

“…Transition metals chelated by nitrilotriacetic acid (NTA) have been successfully applied for purification (Hochuli et al 1987; Ueda et al 2003) and detection of oligohistidine-tagged proteins (Hart et al 2003; Lata et al 2005), as well as for immobilization on surfaces (Sigal et al 1996; Gershon and Khilko 1995; Schmid et al 1997; Xu et al 2004; Schmitt et al 2000). The hexahistidine tag provides binding sites for three NTA moieties, indeed, multiple NTA moieties into single entities increase the affinity adaptors for oligohistidine-tagged proteins (Lata et al 2005).…”