Fluorescent Dyes, Conjugated Secondaries & Labeling

Chromeo™
Fluorescent Chromeo™ Dyes
and Labeling Kits
Fluorescent Secondary
Antibody Conjugates
Reagents for STEDSuper-resolution Microscopy
Chromeo™ Py-Dyes –Fluorescent Chameleons
Fluorescent Assays

complete tool set for successful fluorescence applications

Active Motif’s Chromeo™ product line provides researcherswith high-quality fluorescent dyes that have superior
fluorescent properties, optimized reagents that havebeen tested and verified to work in various applications,as well as a number of innovative cell biology assays.
Use Chromeo products to simplify the preparation ofhigh-quality images in fluorescence microscopy, orto help you develop your own fluorescent assays.

Fluorescent Dyes and Antibodies – fluorescent labels for bioanalysis
Chromeo™ Fluorescent Dyes
Active Motif’s Chromeo™ Dyes – Chromeo™
488, Chromeo™ 494, Chromeo™ 505,
Chromeo™ 546 and Chromeo™ 642 – are used
to detect biomolecules in a variety of assays.
They are typically used to label biomolecules
covalently in a chemical reaction. To give
you more choices when labeling, Chromeo
Dyes are offered in 6 different formats: asamine-reactive NHS-esters, which are useful
to label amino groups on peptides, proteinsor amino-modified DNA; as azides andalkynes for use in click chemistry; as carboxylic acids, and as biotin and streptavidin
conjugates. For application data and ordering
information for the various dye formats,please visit us at www.activemotif.com/dyes.

For the fluorescent properties of each dye,please see Table 1 on the next page.
For the utmost in convenience, the dyes areavailable already conjugated to high-quality
goat anti-mouse and goat anti-rabbit secondary antibodies (see next page).

Why use Chromeo™ Dyes?
• Brightness – high fluorescent intensity
improves sensitivity• Limited photobleaching – enables
multiple exposures and increased
exposure times• pH stability – great for biological assays• Instrument compatibility – work with
common excitation sources like diodelasers, LEDs, tungsten and xenon lamps• Flexible formats – available as
NHS-Esters, Azides, Alkynes, Carboxylic
Acids, and as Biotin, Streptavidin &secondary antibody conjugates

Fluorescent Labeling Kits
Active Motif’s Chromeo™ Antibody Labeling
Kits make it simple to label your antibodies and proteins with the Chromeo Dye ofyour choice. Each kit provides lyophilized
Chromeo 488, 494, 546 or 642 Dye in three

convenient, one-time-use vials that will label
1 mg of antibody/protein each. Also included
are purification columns and the buffers
required for each labeling reaction.

ProductFormat

Catalog No.

Chromeo™ 488 Antibody Labeling Kit

1 kit

15090

Chromeo™ 494 Antibody Labeling Kit

1 kit

15091

Chromeo™ 546 Antibody Labeling Kit

1 kit

15092

Chromeo™ 642 Antibody Labeling Kit

1 kit15093• Easy to use – room temperatureincubations & no harsh chemicals
required for conjugation
Histone H2AX phospho Ser139 pAb+
Chromeo™ 642 anti-rabbit 2°alpha Tubulin mAb+
Chromeo™ 488 anti-mouse 2°

DAPI Nuclear Stain

All 3 images merged

Untreated
cells

Etoposidetreatedcells

Figure 1: Concurrent staining using Chromeo 488 and Chromeo 642 fluorescent secondaries in untreated and etoposide-treated HeLa cells.
HeLa cells were either left untreated, or treated with 100 µM etoposide for 6 hours, prior to fixation with methanol. The histone variant H2AX was then stained withHistone H2AX phospho Ser139 rabbit pAb (Cat. No. 39117) and Chromeo 642 goat anti-rabbit IgG (Cat. No. 15044), while tubulin was visualized using alpha Tubulin mousemAb (Cat. No. 39527) and Chromeo 488 goat anti-mouse IgG (Cat. No. 15031). The nuclei were stained using DAPI and the three separate images were merged.

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Fluorescent secondary antibodies
To help ensure you get the best quality fluorescence microscopy images possible, Active
Motif offers high-quality goat anti-mouseand goat anti-rabbit secondary antibodies
that have been conjugated to a wide variety

of fluorescent dyes, including Chromeo™
Dyes. In addition, we offer ATTO conjugatesthat have been maximally cross-adsorbed
against the IgG’s of a variety of species; thishelps to eliminate background caused bynon-specific binding. Because of the wide
range of dyes offered (see Table 1), you can
use multiple secondaries at the same time
for multi-color staining (see Figure 1, previouspage). HRP conjugates are also available.

Why use Active Motif secondaries?
For fluorescent detection of primary antibodies, it is critical that the secondary antibody bind whether the cells are treated withmethanol, formaldehyde or even formalin.
Active Motif’s fluorescent secondaries havebeen tested and shown to work with highefficiency and specificity under all of themost commonly used fixation conditions.

In addition to the spectral properties of thedye and the quality of the secondary antibody, the quality of a fluorescent conjugate
is influenced by the dye-to protein ratio, theconjugation method and its purity. Active
Motif’s optimized conjugation method,
coupled with subsequent purification of theconjugate from interfering unbound dye

molecules, makes our fluorescent secondaries brighter than other commercially available conjugates and lowers the backgroundin many applications. To ensure your success,Active Motif antibody conjugates have beentested in various applications including flow
cytometry and widefield or confocal fluorescence microscopy.

e L/(mol-cm) Stokes Shift

Dye

Absorption

Emission

Chromeo™ 488

498 nm524 nm73,00026 nm

Chromeo™ 494

489 nm624 nm55,000135 nmChromeo™ 505

514 nm

530 nm70,00016 nm

Chromeo™ 546

550 nm

567 nm

98,80017 nm

Chromeo™ 642

647 nm

666 nm180,000

19 nm

ATTO 594

601 nm

627 nm

120,000

26 nm

ATTO 647N (STED)

644 nm

669 nm150,000

25 nm

ATTO 655 (STED)

663 nm

684 nm

125,000

21 nm

Table 1: Fluorescent properties of dyes when conjugated to secondary antibodies.
All dyes are offered conjugated to both Goat anti-mouse IgG and Goat anti-rabbit IgG
secondary antibodies in regular and sample sizes. HRP conjugates are also available.
Please visit www.activemotif.com/secondary for details and ordering information.

Figure 2: Chromeo 494 staining in HeLa cells.
HeLa cells were stained with rabbit alpha Tubulin polyclonal antibody andChromeo 494 Goat anti-rabbit IgG (Cat. No. 15042) using MAX Stain reagents.

MAX Stain™ Immunofluorescence Tools
MAX Stain™ Immunofluorescence Tools eliminate the challenge of getting high-quality fluorescent images consistently by providing acomplete set of optimized reagents. MAX Stain reagents are used in our daily work to improve the quality of our experiments (Figure 2).
• MAXblock™ Blocking Medium is a non-mammalian, protein-based blocking agent for use in immunofluorescence andimmunohistochemistry. It effectively blocks non-specific antibody binding without reducing signal intensity or specificity.
• MAXwash™ Washing Medium includes a non-mammalian
incubation agent that promotes superior antibody binding
while eliminating non-specific primary and secondary
antibody binding.
• MAXbind™ Staining Medium is a proprietary formulation
designed and optimized to increase antibody binding
specificity and intensity of your IF experiments.

ProductFormatCatalog No.

MAXpack™ Immunostaining Media Kit(includes one each of 15252, 15253, & 15254)

1 kit

15251

MAXblock™ Blocking Medium

150 ml

15252

MAXbind™ Staining Medium

250 ml

15253

MAXwash™ Washing Medium

1000 ml

15254

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Fluorescent Dyes and Antibodies – fluorescent labels for bioanalysis
Get 12-fold greater resolution with STimulated Emission Depletion (STED) microscopy
The Abbe Law of Diffraction LimitingResolution restricts the ability of classicalconfocal microscopy to visually resolve
objects separated by less than ~200 nm. Leica
Microsystems’ STimulated Emission Depletion(STED) microscopy, however, improves resolution up to 12-fold, enabling separation ofsub-cellular structures that previously could

not be resolved. To help ensure your successin STED microscopy, Active Motif collaborated with Leica to create a kit that optimizes
the preparation of samples for STED. In
addition, Leica has tested and certified anumber of Active Motif’s fluorescent dyes.
For more details on our STED products,
please visit us at www.activemotif.com/sted.

Optimized sample preparation for STED microscopy
Proper sample preparation is perhaps themost significant factor for obtaining clear,conclusive, high-resolution images when
performing STED microscopy. To help ensurethat you consistently achieve the best results
possible, Active Motif worked with Leica
Microsystems to develop the Chromeo™
STED Immunofluorescence System. This kit
helps take the guesswork and challenge outof preparing samples for STED microscopy
by providing a complete set of proven,
QC-tested reagents and an optimized protocol. Using the kit is an inexpensive way to becertain you obtain the most scientific value
from the investment in your STED microscope, and the reason Leica recommends thekit for use with its STED microscopes.

Fluorescent dyes recommended by Leica for use in STED
In collaborating to help create the Chromeo™ STED Immunofluorescence System, Leica tested
and now recommends the following Active Motif fluorescent dyes and conjugates for STED:• Chromeo 488 dyes and secondary antibody conjugates are recommended for use with theLeica TCS STED CW microscope, which contains a continuous argon gas laser for excitation.
• Active Motif’s secondary conjugates of ATTO 647N and ATTO 655 (STED) have been certified by Leica for use with the 640 nm laser of the Leica TCS STED microscope.

Figure 3: Confocal and STED microscopy of preand post-synaptic marker proteins.
The localization of pre- and post-synaptic marker
proteins visualized in the dendrites of nerve cells
using conventional, confocal (top image) and STED
microscopy (bottom image). The pre-synaptic
protein Bassoon was stained by ATTO 647N (red),
while the post-synaptic protein Homer was stainedwith Chromeo 494 (green). Images courtesy ofDr. W. Zuschratter, IfN Magdeburg, Germany.
1.0Absorption
Emission

0.8• Chromeo 494 dye and secondary antibody conjugates meet the specifications of anupgraded TCS STED microscope that includes a second excitation laser of 531 nm, enablingdual-color, high-resolution microscopy (Figure 3). The unique fluorescent properties of
Chromeo 494, such as its large Stokes shift (Figure 4), enable you to perform co-localization
studies at resolutions below the diffraction limit. This new method makes it possible toview the nuclear architecture or structures at the synapses in more detail, which will help inthe study and discovery of sub-cellular structures and processes.

0.60.40.20400450500

550600650

Wavelength (nm)700750800

Figure 4: Absorption/Emission of Chromeo™ 494.

Effective fluorescent labels for Click chemistry
Click chemistry constitutes a novel approach for easy, effective labeling and detection of biomolecules in vitro and in vivo. It isbased on the use of biologically unique tags as reaction partners. The use of azide and alkyne tags for labeling proteins, nucleicacids, lipids or sugars has the advantage that neither moiety occurs in nature, and neither reacts with other molecules in cellularsystems. Because of its high selectivity, bio-orthogonal click chemistry can be used for detection in complex biological samples:one of the reaction partners is integrated into the molecule of interest, while the second tag containing a fluorescent label iscovalently attached by performing the click reaction. Chromeo 488, Chromeo 494, Chromeo 546 and Chromeo 642 Dyes areoffered as click-reactive azides and alkynes for use in this highly specific labeling technology.

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Chromeo™ Py-Dyes – simple-to-use chameleon dyes for effective protein labeling
Chromeo™ Py-Dyes are a unique class ofamine-reactive, fluorescent labels with anumber of properties that make them ideal
for labeling proteins. Unlike most otherlabels, Py-Dyes undergo significant changes
in their spectral properties when conjugatedto protein. Upon binding to primary aminegroups, Py-Dyes exhibit a shortwave spectral

shift as well as a large increase in quantum
yield; consequently, only the conjugates emit
detectable fluorescence (Table 2 & Figure 5).
In addition, because unbound Py-Dye ishydrolyzed during the labeling procedure,
there is no need for purification following
the simple one-step, room-temperatureincubation. Labeled protein is ready to use

Py-Dye applications

Why label with Py-Dyes?

Because of their unique properties andadvantages, Py-Dyes can be used inmany different applications:• Broad Stokes shifts and the automatic
degradation of unbound dye during
conjugation eliminate background• Pre- and Post-stain in protein
gel-electrophoresis• Large increase in quantum yield uponbinding causes the non-fluorescent dye
to become fluorescent• Monitoring of amino bonds on surfaces• No change to the net charge of theprotein after labeling• Stain in capillary electrophoresis (CE)• Receptor binding studies• Dye is supplied ready to label in a fast,
simple procedure with no wash steps, orneed to purify the conjugated protein• Protein Quantification• Stain in capillary iso-electric
focusing (CIEF)

Protein(CH2)4

immediately, and any possible backgroundfrom unbound dye is eliminated. Finally, thepositive charge of the amine group is maintained after Py-Dye conjugation, so labeledprotein maintains the same ionic character as
the native protein (Figure 6).

CH3

NH3

Figure 5: Spectral shift of Py-Dyes following
conjugation to protein.
The emission of Chromeo P503 in its unbound,free state is blue. Following conjugation to protein,
the color of the dye changes to red and any
unbound dye automatically degrades.

CH3
RO

Protein(CH2)4

CH3

NR

CH3

Figure 6: Labeling with Py-Dyes maintains each protein’s natural charge.
Chemical equation depicting the chemistry of Py-Dye labeling, which demonstrates the preservation of the positive
charge of the lysine residue of a protein. R is the respective chromogenic/fluorogenic group.

DyeDye State Absorption

Emissione L/(mol-cm)

Quantum
Yield*

Chromeo™ P465

Unbound
Conjugated

645 nm
465 nm732 nm
630 nm25,00025,000Chromeo™ P503

Unbound
Conjugated

612 nm503 nm

665 nm
600 nmChromeo™ P540

Unbound
Conjugated587 nm533 nmChromeo™ P429

Unbound
Conjugated

Chromeo™ P543

Unbound
Conjugated

ProductFormat

Catalog No.

< 1%~14%Chromeo™ P465

1 mg5 mg

15105
16105

60,00024,000< 1%~50%

Chromeo™ P503

1 mg
5 mg

15106
16106

–
627 nm80,00050,0000%~20%

Chromeo™ P540

1 mg
5 mg

15107
16107

457 nm429 nm–
536 nm65,00075,0000%~10%

Chromeo™ P429

1 mg
5 mg

15108
16108

570 nm543 nm–
590 nm110,000
57,0000%~15%

Chromeo™ P543

1 mg
5 mg

15109
16109

Table 2: Properties of Chromeo Py-Dyes.
*Quantum Yield will depend on the Dye-to-Protein Ratio (DPR) of the conjugated protein.

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www.activemotif.com

Albumin Blue Fluorescent Assay Kit

Why use Albumin Blue?
• Quick and easy to use

Bound dyeabsorb (590 nm)
emit (620 nm)

0.6+,'''

0.4Free dye
absorb
emit

0.2

0500

• Unmatched precision

Each kit contains dye for 250 reactions in
96-well plates; it can also be used in smalleror larger formats, such as 384-well plates orcuvettes. The kit also includes human serum
albumin (HSA) to make standard curves forquantifying the albumin in your samples(Figure 8). As albumin is highly conserved, thekit can detect albumin from various species.

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Other assays for determining low albumin
concentrations (< 100 mg/L) are non-specific,
and merely quantify total protein. In contrast, Active Motif’s Albumin Assay Kit issensitive and specific for albumin. Simplyadd the Diluent Buffer and Dye Reagent tothe standards and samples, then read thefluorescence. The intensity of the fluorescent signal is directly proportional to thealbumin concentration of the sample. And,
as albumin-bound dye has a greatly increasedexcitation, the background caused by theemission of any free dye is minimal (Figure 7).

Fluorescence excitation

The Albumin Blue Fluorescent Assay Kit isa quantitative assay for measuring albumin
levels in biological samples, such as serumand urine. The kit combines unmatched sensitivity, high specificity and a fast protocol toprovide an effective way to study albumin.

550600650

700750• Highly selective – no interference fromother proteins or lipids• High-throughput compatible

Figure 7: Absorption and emission spectra of dyethat is free or bound to albumin.
Normalized absorption and emission spectra of free(copper curves) and conjugated dye (purple curves) in
the Albumin Blue Assay.

*,'''*''''),''')''''(,'''('''','''

Wavelength (nm)• Sensitive detection limit (< 0.5 mg/L inurine and serum samples)p4(0-%',o$-.-%+,i)4'%00(+'''''','(''(,')''),'?J8JkXe[Xi[«^&dcFigure 8: Human Serum Albumin standard curve.
An HSA standard curve generated using the control
included in the Albumin Blue Fluorescent Assay Kit.

ProductFormat

Catalog No.

Albumin Blue Fluorescent Assay Kit

1 kit

15002

Cell and Organelle Stains
Active Motif offers several products to study cellular structure using fluorescence microscopy or high-content applications. Our Cell and Organelle Stains
are a collection of innovative fluorescent dyes that provide specific and efficient staining of subcellular structures in live and in fixed cells. A key feature isthat the dyes become fluorescent only after specific binding to target structures; this eliminates background fluorescence caused by any unbound dye.
• LavaCell™ is a naturally fluorescent compound that diffuses into
live or fixed cells, where it stains the plasma membrane andinternal membranes, such as those of the nucleus, Golgi and ER.
• Chromeo™ Live Cell Mitochondrial Staining Kit is a membrane
permeable dye that selectively stains mitochondria in living cells.
The low toxicity and the retention within the cell and thephotostability makes this dye an ideal tool for long-termlabeling of cells and cellular tracking.
Product• Chromeo™ Red Fluorescent Fixed Cell Staining Kit serves asa counterstain in immunofluorescence experiments where
its red color can be used in combination with green dyes
such as Chromeo™ 488, FITC or other 488-excitable dyes.

Figure 9: Live cell staining with Active Motif Cell and Organelle Stains.
On the left: LavaCell stains the plasma membrane and internal membranes ofCHO cells. On the right: Staining of mitochondria (red) and the nuclei (blue) inliving HeLa cells with the Mitochondrial Dye and Hoechst nuclear stain that areincluded in the Chromeo Live Cell Mitochondrial Staining Kit.

FormatCatalog No.

LavaCell™ Live Cell Membrane Staining Kit

200 µg15004

Chromeo™ Live Cell Mitochondrial Staining Kit

1 kit

15005

Chromeo™ Red Fluorescent Fixed Cell Staining Kit

1 kit

15006

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