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HTRF basics.

Published byClement Erick Cummings Modified over 8 years ago

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Presentation on theme: "HTRF basics."— Presentation transcript:

1 HTRF basics

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

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

4 Mithras LB 940 Reagent tubings Waste outlet mains Reagent injectors

5 Time-Resolved Fluorescence

6 Time-Resolved Fluorescence
Lanthanide Chelates Europium Samarium Terbium Dysprosium nm

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

8 Time-Resolved Fluorescence
Different emission wavelengths

9 Time-Resolved Fluorescence
Different emission decay times

10 Time-Resolved Fluorescence
Large Stoke´s shift

11 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

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

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

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

15 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)

16 TRF in Mithras no bottom fluorescence!

17 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 %

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

19 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

20 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

21 Mithras UV/TRF flash filter wheel

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

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

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

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

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

27 Label dialog TRF: HTRF

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

29 Label dialog TRF: HTRF

30 HTRF – further settings
Ratio calculation & HTRF ratio calculation

31 HTRF – further settings
Ratio calculation & HTRF ratio calculation

32 HTRF – further settings
Calculated data are visible in Result pages

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

34 HTRF – export of calculations

35 HTRF – calc. reader evaluat.

36 HTRF – calc. reader evaluat.

37 HTRF – calc. reader evaluat.

38 HTRF – calc. reader evaluat.

39 HTRF – calc. reader evaluat.

40 HTRF – calc. reader evaluat.

41 HTRF – calc. reader evaluat.

42 HTRF – calc. reader evaluat.

43 HTRF – calc. reader evaluat.

44 HTRF – calc. reader evaluat.

45 HTRF – calc. reader evaluat.

46 HTRF – calc. reader evaluat.

47 HTRF – calc. reader evaluat.

48 Applications DELFIA

49 Applications DELFIA

50 Applications DELFIA

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

52 Applications LANCE

53 Applications LANCE

54 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

55 Applications HTRF

56 Applications HTRF

57 Applications HTRF: Cytokines

58 Applications HTRF:

59 Applications HTRF:

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