QDot biolabels talk from invitrogen

QDot biolabels talk from invitrogen - Qdot Conjugates:...

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Qdot ® Conjugates: Sensitive, Multicolor, Stable Fluorescence Patricia Whaley, Ph.D. Molecular Probes Labeling and Detection Technologies Invitrogen Corporation
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2 Outline • Quantum Dot Basics –M a t e r i a l s – Spectral Properties • Microscopy Applications – Immunofluorescent Cell Biology – Molecular Pathology – Cellular Assays •F l o w C y t o m e t r y •I n - v i v o i m a g i n g • Western Blotting
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3 What Are Quantum Dots? 655 605 585 565 525 nm 25nm Size of the nanocrystal determines the color Size is tunable from ~2-10 nm (±3%) Size distribution determines the spectral width Highly fluorescent, nanometer-size, single crystals of semiconductor materials
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4 Qdot Conjugates are Engineered Core Nanocrystal (CdSe) - Determines color Inorganic Shell (ZnS) – Improves brightness and stability Organic Coating - Provides water solubility and functional groups for conjugation Biomolecule - Covalently attached to polymer shell - Immuoglobulins - Streptavidin, Protein A - Receptor ligands - Oligonucleotides 15 - 18 nm
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5 Size of Qdot® Nanocrystals Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA QD 525 QD 605 QD 585 QD 565 QD 655 5nm + 20 nm Gold ~4nm ~5nm ~5nm ~5 x 12nm ~8 x 15nm
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6 Quality Counts—ZStem Shows It Quantum Dot Corp Materials Literature Methods Data courtesy Steve Penneycook (ORNL), James McBride, and Sandy Rosenthal (Vanderbilt)
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7 Qdot Conjugates vs. Competitors Not Created Equal: Immunosorbent Results 0 500 1000 1500 2000 2500 3000 3500 4000 4500 - 0 . 5 0 0 . 0 00 . 5 01 . 0 . 5 02 . 0 . 5 0 Log (Conj Conc nM) RFU on Plate Reader Qdot 605 Anti-Rabbit Lot 1 Qdot 605 Anti-Rabbit Lot 2 Competitor 600 (Fort Orange) Anti-Rabbit 10x competitor data NO measurable binding Clear Dose-Response
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8 Large “Stokes shift” Single-source excitation Narrow emission Excellent photostability Small Stokes shift Multiple source excitation req’d. Broad emission Poor photostability Qdot ® Conjugate (525) Organic dye (FITC) Spectral Properties
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9 Excitation Spectra of Qdot Conjugates High extinction >> High brightness All colors can be excited at the same wavelength, 425DF45 0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06 3.0E+06 3.5E+06 4.0E+06 4.5E+06 5.0E+06 350 450 550 650 750 850 Wavelength (nm) Qdot 525 Conjugate Absorbance Qdot 565 Conjugate Absorbance Qdot 585 Conjugate Absorbance Qdot 605 Conjugate Absorbance Qdot 655 Conjugate Absorbance Qdot 705 Conjugate Absorbance Qdot 800 Conjugate Absorbance Organic Dyes {
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10 Emission Spectra of Qdot Conjugates 400 500 600 700 800 900 Wavelength (nm) 525 525 605 605 655 655 705 705 800 800 565 565 Minimal (<5%) cross-talk using 20nm bandpass filters Simplified signal un-mixing >> simplified multiplex labeling
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11 Exp. Time: 0.44 seconds 8.12 seconds Exp. Time: 0.019 seconds 1.22 seconds High level Her 2/neu expression in SK-BR-3 Cells Quantum dots up to 50x brighter Low level of Her 2/neu expression in MDA-MB- 231 cells Quantum dots easy to detect but dye undetectable Excellent Brightness Provides High Sensitivity Quantum dot Organic dye
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12 525 (Mouse) Mitochondria 565 (Rat) Tubulin 605 (Rabbit) Ki-67 655 (Human) Nuclear antigen 705 (Streptavidin) Actin
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This document was uploaded on 01/03/2012.

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QDot biolabels talk from invitrogen - Qdot Conjugates:...

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