Fluorescent principle
Fluorescent microscope
DAPI
345
455
FITC
495
519
Alexa 488
499
519
Tetramethylrhodamine/TRITC
551
577
Rhodamin
558
574
Rhodamin Red-X
571
at 543: 33%
591
Alexa 568
577
at 543: 40 %
603
Alexa 647
653
669
Texas-Red and Texas-Red-X
589
556
at 543: 40%
615
Alexa 594
590
at 543: 38 %
618
489
553
651
Cy-Series
506
575
674
GFP and GFPuv
489
558
E-GFP's and
DsRed
506
583
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
Structure of GFP
• 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
GFP-Fluorophore
• Fluorescence is
mediated by a
fluorophore located at
the center of the
protein
Structure of the GFP-fluorophore
• The fluorophore is
derived from the
tripeptide -Ser65-Tyr66Gly67• Formation of the
fluorophore occurs in
an autocatalytic
manner
Light sources for Fluorescence
Filtersets
and GFP's
Quantum efficiency of Chip
spectrum of mercury lamps
Quantum efficiency of chip
New Filterset
New Filterset-other
flourochromes
Old Filtersets
Old filtersets- other flurochromes
Antibodies used for fluorescence
Li st o f recomm ende d an tibo dies use d fo r imm unfl uores cen ce
Ab sorbtion
Emi sssi on
E-GFP
48 9
50 8
Al exa 48 8
49 9
51 9
an ti-Maus
AK1 90
an ti-Rat
AK1 84
an ti-Rabb it
AK1 87
Cy3
52 5/55 3
57 5
Rh-Red-X
54 0/57 1
59 1
Al exa 56 8
54 5/57 7
60 3
AK1 83
Rhod amin
55 8
57 4
AKO57
AK1 98
AK0 43 o r AK05 3
Th e choice o f th e fl ourochrom e:
tri ple-l abel ling
Al exa 48 8 fo r gree n si gnal
Rh-Red-x o r Ale xa 568 for red s igna l
Cy5 fo r infrared
Cy3 will be the best flu orochrome for the red chan nel but it wil l al so s how up weakly in the green ch anne l
Tx-Red is n ot g ood for the n ew set of filters
Rhom dami n mi ght be o .k.
Tx-Red
55 6/58 9
61 5
AK2 02
AK1 54
AKOO2
Cy5 / Alexa 647
61 2/65 3
66 9
AK0 48 o r AK04 9
AK1 69
AK1 82
Laser
and
Fluorochromes