A Guide to Fluorochromes - Abcam

1 A Guide to FluorochromesDiscover more 2012 Abcam , All Rights Reserved. The Abcam logo is a registered trademark. All information/detail is correct at time of going to print. Color bars are an approximation of corresponding emission is a trademark of Thermo Fisher Scientific and its subsidiaries. Cy and CyDye are trademarks of GE Healthcare Limited. Alexa Fluor is a trademark of Molecular Probes Inc. HILYTE FLUOR is a trademark of AnaSpec, Inc. DRAQ5 , DRAQ7 and CyTRAK Orange are trademarks of Biostatus excitation Excitation laser wavelength (nm)wavelength (nm)lines (nm)Methoxycoumarin360410 DyLight 405400420 Alexa Fluor 405402421 Brilliant Violet 421 407421 HiLyte FluorTM 405404428 DyLight 350353432 Alexa Fluor 350346442 Aminocoumarin (AMCA)350445BD Horizon V450404448 Pacific BlueTM404456360,405,407 EviTagTM quantum dots-Lake Placid Blue470490 AMCyan457491BD Horizon V500415500Cy2 489506488 ChromeoTM 488488517 DyLight 488493518 Alexa Fluor 488495519488 FAM494519 Fluorescein Iso-thiocyanate (FITC)

2 495519488 EviTagTM quantum dots-Adirondack Green505520 ChromeoTM 505505526 HiLyte FluorTM 488501527 Alexa Fluor 514518540 EviTagTM quantum dots-Catskill Green525540 Alexa Fluor 430434541 Pacific Orange 403551 Alexa Fluor 532532554 HEX535556 EviTagTM quantum dots-Hops Yellow545560 ChromeoTM 546545561Cy3 548561488,514 Alexa Fluor 555555565 HiLyte FluorTM 5555505665-TAMRA541568 Alexa Fluor 546556573532 DyLight 550562576 Phycoerythrin (PE)496,566576488 Tetramethyl Rhodamine Isothiocyanate (TRITC)557576 EviTagTM quantum dots-Birch 576589568,543 Rhodamine Red-X570590PE-Dyomics 590488599 EviTagTM quantum dots-Fort Orange585600 ROX575602 Alexa Fluor 568578603532 Red 613480,565613 Texas Red 595613568,543,514 HiLyte FluorTM 594593616PE-Texas Red 566616 Alexa Fluor 594590617 DyLight 594593618 EviTagTM quantum dots-Maple-Red Orange600620 Alexa Fluor 610612628 ChromeoTM 494494628 Alexa Fluor 633632647 SureLight APC 652657 DyLight 633638658 Allophycocyanin (APC)650660595,633,635,647 ChromeoTM 642642660 Quantum Red488660 SureLight P3614662 Alexa Fluor 647650665595,633,635,647Cy5 647665633,635PE-Cy5 565666488 SureLight P1545666PE-Alexa Fluor 647567669PE-Dyomics 647488672 DyLight 650654673 HiLyte FluorTM 647650675 Peridinin Chlorophyll (PerCP)

3 477678488 IRDye 700DX680687 Alexa Fluor 650694595,633,635, 675694647 TruRed490,675695 HiLyte FluorTM 680678699 Alexa Fluor 680679702 DyLight 680692712 Alexa Fluor 700702723 APC-Cy7 650774595,633,635,647 Alexa Fluor 750749775Cy7 753775PE-Dyomics 747488776 DyLight 755754776 HiLyte FluorTM 750753778PE-Cy7 566778488 IRDye 800RS770786 DyLight 800 777794 IRDye 800CW778794 Alexa Fluor 790782805 Nucleic Acid DyesMax. excitation Max. emission Excitation laserwavelength (nm)wavelength (nm)lines (nm)DAPI359461325,360,405,407 Hoechst 33258352461 Hoechst 33342350461 SYTOX Blue431480 YOYO-1491509 SYTOX Green504523 TOTO-1, TO-PRO-1509533 Mithramycin450570 SYTOX Orange547570 Chromomycin A3445575 CyTRAK Orange *457,488,549615 Ethidium Bromide493620 Propidium iodide (PI)305,540620325,360,488 DRAQ5 646681,697 DRAQ7 599, 644678,697 Single dyeFor a single fluorescent dye, a laser set at the signature excitation wavelength for the dye provideselectromagnetic energy to an electron in that molecule(1).

4 The electron moves to an excitation state at thenext energy level (Es)(2). Energy is then released in the form of a photon (fluorescence) and the electronmoves back down to the lower energy level (Gs)(3). In the case of atandem fluorescent dye, after excitationof the electron by a laser (1 - 2), energy is released by an electron in the donor molecule(D) and absorbedby an electron in the acceptor molecule (A) (4). The electron in the acceptor molecule moves to an excitationstate at the next energy level (Es). Similar to a single dye, energy is then released in the form of the photon (fluorescence) and the electron moves back down to the lower energy level (Gs) (5). 800 Relative Intensity 350 400 500 550 600 650 700 750 800 450 Wavelength (nanometers) Excitation Emission PE Laser line (488) Single (PE) Relative Intensity 350 400 500 550 600 650 700 750 450 Wavelength (nanometers) Excitation Emission Cy5 Laser line (633) Single (Cy5 ) Relative Intensity 350 400 500 550 600 650 700 750 800 450 Wavelength (nanometers) Excitation Emission PE-Cy5 Laser line (488) Tandem (PE-Cy5 ) Excitation and Emission Spectral ProfilesEnergy Level Diagrams Fluorochromes have unique and characteristic spectra for absorption (excitation) and emission.

5 A single dyeis excited at a particular wavelength and emits a photon at a higher wavelength. A tandem dye consists of adonor and acceptor fluorochrome molecule, placed in close proximity, allowing for energy transfer betweenthe two. The tandem dye is excited at the excitation wavelength of the donor molecule and emits a photon atthe emission wavelength of the acceptor molecule. EsGsExcitationFluorescence(Emission)Lase r132 EsGsExcitationFluorescence(Emission)Lase r12AD43 Tandem dye2 DRAQ5 (ab108410) allows rapid staining ofdsDNA/nuclei of LIVE or FIXED cells. It isideal for use in multi-color analysis andcompatible with GFP labels. As well asoffering half the scan times of Hoechst orDAPI, DRAQ5 can be used in a greatervariety of applications. The image on the leftshows HeLa cells stained with 5 M DRAQ5 to label nuclear DNA (pseudocolor orange).

6 Pre-absorbed goat polyclonal secondaryantibodies to rabbit IgG - H&L were used tovisualise beta Catenin (DyLight 488,ab96899, green) and alpha Tubulin (DyLight 594, ab96899, red).1123A3D4 Multicolor staining exampleMERGEDab16051 - Beta CateninDRAQ5ab7291 - Alpha Tubulin*CyTRAK Orange stains both nucleus and cytoplasm148_12_AKR Fluorochromes Poster02:Layout 1 23/04/2012 15:10 Page 1