Fluorescent Dyes and Assays for Genomics and ... - Interchim

Safer, ultra-sensitive alternativesto EtBr and SYBR® dyesFEATURESSafer than EtBrShown by Ames test and other tests to benonmutagenic and noncytotoxic.Number of Colonies1000750500250SYBR GreenEtBrGelGreenGelRedEasy disposalPassed environmental safety tests for directdisposal down the drain or in regular trash.Ultra-sensitiveMore sensitive than EtBr and SYBR ® Safe.Extremely stableStable in solution at room temperature.Simple to useFor precast or post-electrophoresis gel staining.Compatible with standard instrumentsGelRed replaces EtBr; GelGreen replacesSYBR ® dyes.00 0.1 0.5 1 5 10 25 50Dose (ug/plate)Figure 4. GelRed and GelGreen are non-mutagenic. Comparison of mutagenicityamong GelGreen, GelRed, SYBR ® Green I and EtBr in +1 frameshift Salmonellaindicator strain TA98 in the presence of S9 fraction. SYBR® Green I became cytotoxic at50 ug/plate. For more information, download the GelRed and GelGreen Safety Reportat www.biotium.com.Table 1. Ordering InformationDescription Cat. No. Unit Size FeaturesGelRed10,000X in waterGelRed10,000X in DMSO41003-T 25 uL41003 0.5 mL41003-1 10 mL41002 0.5 mLNewer, saferformulation in water(recommended fornew users)Original formulation inDMSOGelRed3X in water41001 4 LReady to use forpost-stainingSYBR® Safe GelRed GelGreenGelGreen10,000X in waterGelGreen10,000X in DMSO41005-T 25 uL41005 0.5 mL41005-1 10 mL41004 0.5 mLNewer, saferformulation in water(recommended fornew users)Original formulation inDMSOReady-To-Use1 kb DNA LadderReady-To-Use100 bp DNA Ladder3102231032150 lanes150 lanesOptimal concentrationfor GelRed/GelGreen precastgels*1 kb DNA Ladder 31021 30 ug100 bp DNA Ladder 31031 30 ugStand-alone laddersFigure 3. GelRed and GelGreen gel stains are safer because they cannotpenetrate cell membranes to bind DNA in living cells. HeLa cells were incubatedat 37 o C with 1X SYBR® Safe, GelGreen or GelRed, respectively. Imageswere taken following incubation with dye for 30 min. The top row shows phasecontrast images of the field of cells, the bottom row shows green fluorescence forSYBR® Safe and GelGreen and red fluorescence for GelRed. SYBR® Saferapidly entered cells and stained nuclei. GelRed and GelGreen were unableto cross cell membranes, demonstrated by the absence of fluorescence staining.DNA Gel Extraction Kit5X TBE Buffer 41006 4 L31030-50 50 columns Validated for usewith GelRed and31030-250 250 columns GelGreen*Electrophoresis bufferconcentrate* GelRed and GelGreen also are compatible with popular gel extraction kitsand DNA ladders from other suppliers.Genomics and Proteomics Products • www.biotium.com • 800-304-5357 3

Superior sensitivity and broad dynamic rangeFluorescence120010008006004002000dsDNA-Selective AssaysdsDNAFigure 2: Triplicate samples of calf thymus dsDNA, mouse liver RNA orsalmon sperm ssDNA were assayed with the AccuBlue High SensitivitydsDNA Quantitation Kit. The AccuBlue High Sensitivity Assay is selectivefor double-stranded DNA over RNA or single-stranded DNA.AccuBlue Broad Range vs. Quant-iT Broad Range (2-4000 ng)RNAssDNA0 20 40 60 80 100DNA/RNA (ng/well)FluorescenceAccuBlue Broad Range (2-2000 ng)70006000500040003000200010000Fluorescence75502500 5 10 15 20DNA (ng/well)R² = 0.99790 500 1000 1500 2000DNA (ng/well)Figure 3: Standard curve of calf thymus DNA assayed using AccuBlue BroadRange Kit and read on a microplate reader (Ex/Em 350/460). Inset showsthe lower end of the titration.AccuBlue High Sensitivity (0.2-100 ng)Fluorescence900060003000AccuBlueQuant-iTFluorescence2500200015001000500Fluorescence10075502500 1 2 3 4 5DNA (ng/well)R² = 0.999400 1000 2000 3000 4000DNA (ng/well)Figure 4: Two-fold dilutions of calf thymus DNA were assayed using AccuBlueor Quant-iT Broad Range assay kits. AccuBlue has improved linearity andwider dynamic range than the Quant-iT Broad Range.00 20 40 60 80 100DNA (ng/well)Figure 5: Standard curve of calf thymus DNA assayed using the AccuBlueHigh Sensitivity Kit and read on a microplate reader (Ex/Em 485/530). Insetshows the lower end of the titration.AccuClear Ultra High Sensitivity 0.03-250 ng)Fluoresence140001200010000800060004000Fluoresence60402000 0.2 0.4 0.6 0.8 1R² = 0.998DNA ng/wellAccuBlueHS DyePicoGreen Dye200000 50 100 150 200 250DNA ng/wellFigure 6: Standard curve of calf thymus DNA assayed using the AccuClearUltra High Sensitivity Kit and read on a microplate reader (Ex/Em 460/507).Inset shows the lower end of the titration.Figure 7: The AccuBlue High Sensitivity (HS) reagent and PicoGreen were dilutedto working concentrations in HeLa cell cultures and incubated for 30 minutes.PicoGreen and the Quant-iT High Sensitivity reagent (Invitrogen) readily bindnuclear DNA while no nuclear staining is evident with the AccuBlue HS reagent,illustrating its overall safety and lower toxicity due to its membrane impermeability.Ordering InformationLinear Range Description Cat. No. Unit SizeBroad Range2-2000 ngHigh Sensitivity0.2-100 ngUltra HighSensitivity0.03-250 ngAccuLite MiniFluorometersAccuBlue Broad Range dsDNA Quantitation Solution, Trial Size 31009-T Each, 200 AssaysAccuBlue Broad Range dsDNA Quantitation Solution 31009 Each, 1000 AssaysAccuBlue Broad Range dsDNA Quantitation Kit with 9 DNA standards 31007 Each, 1000 AssaysAccuBlue High Sensitivity dsDNA Quantitation Solution, Trial Size 31008-T Each, 200 AssaysAccuBlue High Sensitivity dsDNA Quantitation Solution 31008 Each, 1000 AssaysAccuBlue High Sensitivity dsDNA Quantitation Kit with 8 DNA Standards 31006 Each, 1000 AssaysAccuClear Ultra High Sensitivity dsDNA Quantitation Solution, Trial Size 31027-T Each, 200 AssaysAccuClear Ultra High Sensitivity dsDNA Quantitation Solution 31027 Each, 1000 AssaysAccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 7 DNA Standards 31028 Each, 1000 AssaysAccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard 31029 Each, 4000 AssaysAccuLite 350 Mini Fluorometer E90000 EachAccuLite 470 Mini Fluorometer E90001 EachGenomics and Proteomics Products • www.biotium.com • 800-304-5357 9

AccuOrange Protein Quantitation KitHighly sensitive, fluorometric protein assayAccuOrange Protein Quantitation Kit is a highly sensitive fluorescence-based assay for quantitating purified protein samples in 96-well format. The detectionrange of the assay is 0.1-15 ug/mL protein. AccuOrange is much more sensitive than traditional protein quantitation assays such as BCA, Bradford and Lowry,and shows superior linearity and reproducibility compared to the NanoOrange® protein quantitation assay (Figure 2). The assay shows minimal variabilitybetween different proteins, and has stable fluorescence signal for up to 16 hours.AccuOrange is recommended for quantitating purified protein or antibody samples. The tolerance of the AccuOrange assay to salts, buffers, detergents, andother chemicals is shown in Table 1. The AccuOrange assay has low tolerance for non-ionic detergents, and is not recommended for use with cell lysatescontaining Triton X-100, sodium deoxycholate, CHAPs, or other non-ionic detergents. The assay can tolerate up to 0.01% SDS (final concentration in assay).Table 1. Comparison of AccuOrange with other protein quantitation assays• Ex/Em: 480/598 nmFEATURES• Linear detection range: 0.1-15 ug/mL protein• 200 uL microplate assay• Minimal protein-protein variation• Fluorescence signal stable for at least 16 hours• Compatible with reducing agents, amino acids,nucleic acids, and imidazole• For use with purified protein or antibody samples• Compatible with AccuLite 470 Mini-Fluorometer(see p. 8)Fluorescence2500200015001000500Fluorescence40020000 0.5 1 1.5BSA (ug/mL)NanoOrangeR² = 0.9453AccuOrangeR² = 0.9981AccuOrangeNanoOrange00 5 10 15BSA (ug/mL)Assay TypeAccuOrangeNanoOrange®Modified LowryBCABradford(Coomassie)Pierce® 660 nmA 280Detection Range(microplate assay)0.1-15 ug/mL0.1-10 ug/mL1-1500 ug/mL20-2000 ug/mL50-500 ug/mL50-2000 ug/mL50-2000 ug/mLComments• Fluorescence detection (480/598 nm)• Highly linear• Signal stable for at least 16 hours• Compatible with reducing agents• Not compatible with detergents• Fluorescence detection (470/570 nm)• Non-linear• Fluorescence stable for 6 hours• Compatible with reducing agents• Not compatible with detergents• Absorbance detection (750 nm)• Non-llinear• Not compatible with reducing agents• Not compatible with detergents• Absorbance detection (562 nm)• Highly linear• Signal not stable over time• Not compatible with reducing agents• Compatible with detergents• Absorbance detection (595 nm)• Signal not stable over time• Non-linear• Compatible with reducing agents• Not compatible with detergents• Absorbance detection (660 nm)• Non-linear• Compatible with reducing agents• Compatible with detergents• Absorbance detection (280 nm)• High protein-protein variability• Contaminants such as nucleic acidscan affect resultsNanoOrange is a registered trademark of Molecular Probes Inc.Figure 1. AccuOrange shows better linearity and reproducibility compared to NanoOrange®Protein Quantitation assay. BSA titration was performed in triplicate using AccuOrange ProteinQuantitation Kit or NanoOrange Protein Quantitation Kit from Life Technologies according tomanufacturer’s protocol and read on a microplate reader at the recommended wavelengths foreach assay. Inset shows the lower end of the curve. Error bars represent standard deviation ofthe mean for triplicate samples.Ordering InformationDescription Cat. No. SizeAccuOrange Protein Quantitation Kit30071-T 200 assays30071 2000 assaysGenomics and Proteomics Products • www.biotium.com • 800-304-5357 11

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