HTRF basics.

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Presentation transcript:

HTRF basics

Microplate reader portfolio dedicated multi Absorbance ☑ Luminescence Fluorescence Intensity Fluorescence Polarisation Time-Resolved Fluorescence AlphaScreen BRET

Mithras LB 940 PMT & emission filters Flash excitation lamp Halogen Plate carrier excitation filters

Mithras LB 940 Reagent tubings Waste outlet mains Reagent injectors

Time-Resolved Fluorescence

Time-Resolved Fluorescence Lanthanide Chelates Europium Samarium Terbium Dysprosium Excitation @ 320-340 nm

Time-Resolved Fluorescence Long-lived fluor.: up to 1000 µs

Time-Resolved Fluorescence Different emission wavelengths

Time-Resolved Fluorescence Different emission decay times

Time-Resolved Fluorescence Large Stoke´s shift

Time-Resolved Fluorescence DELFIA® heterogeneous method: unbound labelled molecules must be washed away HTRF® (CisBio); LANCE (PKI) , LanthaScreen (Invitrogen) homogeneous methods: energy transfer between cryptate and acceptor

TRF - DELFIA as immunoassay Signal after re-chelating w/ Enhancement solution Emission @ 615 nm

LANCE: TR-FRET the homogeneous method (PKI) Eu-Chelate as donor (615 nm) APC, Cy5 or ULight as acceptor (665 nm)

HTRF®: TR-FRET the homogeneous method (CisBio) Eu-Cryptate as donor (620 nm) XL665 as acceptor (665 nm)

Time-Resolved Fluorescence Xenon flash lamp broad-band excitation filter: 320 nm (HTRF), 340 nm (DELFIA) confocal emission optics emission filter 615 nm (DELFIA Eu) emission filters 620 nm (Cryptate Eu) and 665 (acceptor e.g. XL665) gated PMT (red enhanced response for TR-FRET)

TRF in Mithras no bottom fluorescence!

HTRF performance CisBio reader evaluation kit (white 96 well half area) Mithras typical target S/N 620 nm (control/bkg) 70 40 cv of ratio 0 Std 5 % 10 % Delta F of Low FRET Std 28 % 15 % Delta F of High FRET Std 900 % 600 %

Mithras TRF performance – sensitiv. 96 well: <6 amol Eu / well 384 well: <2 amol Eu / well

Mithras TRF performance 96 well 384 well Mithras 6 amol 2 amol Victor 10 amol na Envision 12 amol 3 amol Fluo/PolarStar 50 amol PheraStar 1 amol Synergy II Spectramax M5 35 amol Genios Pro 20 amol DTX 880 30 amol Chameleon

Mithras TRF performance - crosstalk 384 well: < 10-6 = not detectable compare Victor: stand. beam: 8 * 10-5 narrow beam: 3 * 10-5 Mithras is 100 times better ! NOTE: high crosstalk affects “real” detection limit

Mithras UV/TRF flash filter wheel

Label dialog TRF: DELFIA Counting time per well Always full lamp energy Emission filter Cycle time: 2000 Delay Time: 400 Reading Time: 400

Label dialog TRF: HTRF Open default HTRF.par file or start from scratch Click Options | Read

Label dialog TRF: HTRF Samples tab: Plate selection Measured wells Injected wells Reading order

Label dialog TRF: HTRF Measurement tab: Operation settings Operation sequence Temperature settings

Label dialog TRF: HTRF Counting time per well Always full lamp energy Emission filter Cycle time: 2000 Delay Time: 50 Reading Time: 300

Label dialog TRF: HTRF

Label dialog TRF: HTRF Export with “RawData Export Driver”

Label dialog TRF: HTRF

HTRF – further settings Ratio calculation & HTRF ratio calculation

HTRF – further settings Ratio calculation & HTRF ratio calculation

HTRF – further settings Calculated data are visible in Result pages

HTRF – export of calculations Export with “Matrix Export Driver”

HTRF – export of calculations

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

HTRF – calc. reader evaluat.

Applications DELFIA

Applications DELFIA

Applications DELFIA

Applications LANCE Kinase cAMP Caspase Helicase Generic reagents for receptor binding, transfer, etc.

Applications LANCE

Applications LANCE

Metabolic Disease & CNS Applications Metabolic Disease & CNS Insulin Cortisol Estradiol Amyloid β1-40 HTRF® GPCR cAMP Cell-Based Assay cAMP Membrane Assay IP-One HTRF Assay cGMP Assay Oncology Ubiquitin assay Heparanase assay Inflammation Cytokines PGE2 Histamine cGMP Kinase HTRF® KinEASE™ Universal Ser/Thr Assays Kinase Toolbox Reagents

Applications HTRF

Applications HTRF

Applications HTRF: Cytokines

Applications HTRF:

Applications HTRF: