Fluorescent principle

Fluorescent microscope

345 455

DAPI

495 519

FITC

499 519

Alexa 488

Tetramethylrhodamine/TRITC

551 577

558 574

Rhodamin

571 591

at 543: 33%

Rhodamin Red-X

577 603

at 543: 40 %

Alexa 568

Alexa 647

653 669

589 615

556

at 543: 40%

Texas-Red and Texas-Red-X

590 618

at 543: 38 %

Alexa 594

553 651489

575 674506

Cy-Series

GFP and GFPuv

583506

558489

E-GFP's and DsRed

Table of Fluorescent Proteins

What is green fluorescent protein (GFP) ?

• GFP is a 238-amino acid protein originally isolated from the bioluminescent jellyfish Aequoria victoria

• Can be expressed in any organism without additional cofactors

• Structurally, GFP represents a new motif which has been named the “β-can”

• The “can” consists of 11 β-strands and a central α-helix

Structure of GFP

GFP-Fluorophore

• Fluorescence is mediated by a fluorophore located at the center of the protein

• The fluorophore is derived from the tripeptide -Ser65-Tyr66-Gly67-

• Formation of the fluorophore occurs in an autocatalytic manner

Structure of the GFP-fluorophore

Light sources for Fluorescence

Filtersetsand GFP's

Quantum efficiency of Chipspectrum of mercury lamps Quantum efficiency of chip

New Filterset

New Filterset-other flourochromes

Old Filtersets

Old filtersets- other flurochromes

Antibodies used for fluorescenceList of recommended antibodies used for immunfluorescence

E-GFP Alexa 488 Cy3 Rh-Red-X Alexa 568 Rhodamin Tx-Red Cy5 / Alexa 647Absorbtion 489 499 525/553 540/571 545/577 558 556/589 612/653Emisssion 508 519 575 591 603 574 615 669

anti-Maus AK190 AK183 AKO57 AK202

anti-Rat AK184 AK198 AK154 AK169

anti-Rabbit AK187 AK043 or AK053 AKOO2 AK048 or AK049 AK182

The choice of the flourochrome:

triple-labelling

Alexa 488 for green signalRh-Red-x or Alexa 568 for red signalCy5 for infrared

Cy3 will be the best fluorochrome for the red channel but it will also show up weakly in the green channelTx-Red is not good for the new set of filtersRhomdamin might be o.k.

Laser and

Fluorochromes