Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels.
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.
Hart
Courtenay
C
Molecular Probes, Eugene, OR 97402, USA.
Schulenberg
Birte
B
Diwu
Zhenjun
Z
Leung
Wai-Yee
WY
Patton
Wayne F
WF
D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D011994:Recombinant Proteins; D006639:Histidine