A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis.
Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon-arc lamps; 488 nm and 457 nm Argon-ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid-state lasers; 543 nm helium-neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one-dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G-250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI-MS and Edman-based sequencing chemistry.
Mackintosh
James A
JA
FLUOROtechnics Pty Ltd., Department of Chemistry, Macquarie University, Sydney, NSW 2109, Australia.
Choi
Hung-Yoon
HY
Bae
Soo-Han
SH
Veal
Duncan A
DA
Bell
Philip J
PJ
Ferrari
Belinda C
BC
Van Dyk
Derek D
DD
Verrills
Nicole M
NM
Paik
Young-Ki
YK
Karuso
Peter
P
D000818:Animals; D001578:Benzopyrans / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes / Q000737:chemistry*; D005658:Fungi / Q000737:chemistry; D005663:Furans / Q000737:chemistry*; D007659:Ketones / Q000737:chemistry*; D051379:Mice; D011506:Proteins / Q000737:chemistry; D051381:Rats; D012394:Rosaniline Dyes / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001578:Benzopyrans; D005456:Fluorescent Dyes; D005663:Furans; D007659:Ketones; D011506:Proteins; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; C477464:epicocconone; C004692:coomassie Brilliant Blue