Secondary Antibodies and Conjugates

92 Pages·2014·1.64 MB·English

F(ab’)2 Fragment Affinity-Purified
Ordering e-mail:
Specialising in
Secondary Antibodies and Conjugates
Immunofluorescence
Western Blotting
Flow Cytometry
In SituHybridization
Electron Microscopy
Enzyme Immunohistochemistry
STED Microscopy
2-photon Microscopy
ELISA
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conjugation of affinity-purified secondary antibodies and purified immunoglobulins. anytime during normal office hours (GMT), which are:-
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Affinity Purified Whole IgG, F(ab')2 Fragment and Monovalent Fab Fragment
Antibodies with a multitude of species and conjugate options.
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There are in addition to our Flow Cytometry and Western Blot optimised antibodies,
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an extensive range of blocking reagents and much more, providing a comprehensive
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Conditions: Technical Assistance
ALL PRODUCTS LISTED IN THIS CATALOGUE ARE FOR IN VITRORESEARCH USE ONLY. No product listed in this catalogue is a medical device. They
are not intended for diagnostic or therapeutic purposes.Nothing disclosed herein is to be construed as a recommendation to use our products in violation of any Our highly qualified and dedicated team
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Contents
Ordering e-mail: [email protected] Technical e-mail: [email protected]
Ordering Information.....................................................................inside front cover Cy2, Cy3 and Cy5 Conjugates for Permanent Mounting.................................68
Technical Information - Affinity-Purified Secondary Antibodies.....................4 Streptavidin...........................................................................................................71
Selection and Location of Affinity-Purified Secondary Antibodies..........................4
Multiple Labelling (ML) Using Conjugated Secondary Antibodies.........................7 ImmunoGold Reagents........................................................................................72
Probes Conjugated to Affinity-Purified Antibodies and to Other Proteins...............8 LM Grade 4 nm Colloidal Gold - Antibody Complexes........................................73
Fluorescent Dyes...........................................................................................8 EM Grade 6 nm, 12 nm & 18 nm Colloidal Gold - Antibody Complexes..............74
Alexa Fluor®.......................................................................................8-12
Cyanine (CyTM2, Cy3, Cy5)...............................................................8-12 Peroxidase-Anti-Peroxidase (PAP) Soluble Immune Complexes.................75
DyLight 405..........................................................................................10
Aminomethylcoumarin Acetate (AMCA).................................................10 Solid-Phase Immunoadsorbent Gels................................................................75
Fluorescein Isothiocyanate (FITC/DTAF)...............................................11
Rhodamine RedTM-X (RRX)...................................................................12 Primary Antibodies for Signal Enhancement...................................................76
Biotin-SPTM(long spacer)............................................................................13 Purified IgG Fraction Monoclonal Mouse Anti-Digoxin........................................76
Enzymes......................................................................................................13 Purified IgG Fraction Monoclonal Mouse Anti-Biotin...........................................76
Purified IgG Fraction Monoclonal Mouse Anti-Fluorescein..................................76
Whole IgG Affinity-Purified Secondary Antibodies....................................14-35 Affinity-Purified Anti-Horseradish Peroxidase......................................................76
F(ab')2 FragmentAffinity-Purified Secondary Antibodies.........................36-45 Normal Serums and Gamma Globulins.............................................................78
Monovalent Fab Fragment Affinity-Purified Secondary Antibodies Bovine Serum Albumin (IgG-Free, Protease-Free)..........................................79
for Blocking and for Double Labelling Primary Antibodies from
the Same Host Species.......................................................................................46-52 Purified Proteins from Normal Serums........................................................80-89
Secondary Antibodies for Flow Cytometry..................................................53-58 Antisera to Immunoglobulins, Whole Serums and Enzymes.........................90
IgG Controls and Streptavidin Conjugates for Flow Cytometry.....................58 Trademarks and Licences...........................................................inside back cover
Anti-Mouse IgG Subclass Specific Antibodies................................................60
Anti-IgG, Light Chain Specific Antibodies for Western Blotting
after Immunoprecipitation (IP)...........................................................................62
Alexa Fluor®680 and Alexa Fluor®790 Conjugates for
High Sensitivity Western Blots...........................................................................64
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Technical Information - Affinity-Purified Secondary Antibodies
Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using
antigens coupled to agarose beads. A proprietary elution process is used to dissociate lgG F(ab’) Fab
H H H 2 H H
antibodies from the antigen. Unconjugatedaffinity-purified antibodies are supplied sterile-filtered L L L L L
in phosphate buffer without stabilisers or preservatives. Conjugatedaffinity-purified antibodies
pepsin over
are freeze-dried in phosphate buffer with stabilisers and sodium azide, with the exception of
horseradish peroxidase conjugates, which do not contain a preservative. Alkaline phosphatase digestion + digestion
conjugates are freeze-dried in Tris buffer with stabilisers and sodium azide. fragments
Selection and Location of Affinity-Purified Secondary
lgG Fab Fc
Antibodies H H
L L H
papain L
+
Step 1. Affinity-purified secondary antibodies are offered in three different
digestion
forms. H H H
Select from Whole IgG (pages 14-35), F(ab')2fragment (pages 36-45), or Fab fragment
(pages 46-52) antibodies. Figure 1.Schematic representation of IgG fragments generated by enzymatic digestions.
Figure 1.
Whole IgG(pages 14-35) antibodies are isolated as intact molecules from antisera by
immunoaffinity chromatography. They have an Fc portion and two antigen binding Fab cells in buffer containing 5% normal serum from the host species of the labelled secondary
portions joined together by disulphide bonds (Figure 1) and therefore they are divalent. The antibody. To prevent capping, endocytosis and regeneration of Fc receptors on living cells,
average molecular weight is reported to be about 160 kDa. The whole IgG form of antibodies incubate at 4°C in buffer containing 5% normal serum with sodium azide added to inhibit
is suitable for the majority of immunodetection procedures and is the most cost effective. metabolism.
F(ab')2fragment(pages 36-45) antibodies are generated by pepsin digestion of whole Caution: Never block with normal serum or IgG from the host species of the primary
IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. antibody. If immunoglobulins in normal serum bind to the specimen of interest, they will be
F(ab')2fragments have two antigen-binding Fab portions linked together by disulphide bonds recognised by the labelled secondary antibody, resulting in higher background.
and therefore they are divalent. The average molecular weight is about 110 kDa. They are
used for specific applications, such as to avoid binding of secondary antibodies to live cells Bovine serum albumin (BSA) and dry milk, both commonly used for blocking, may contain
with Fc receptors or to Protein A or Protein G. bovine IgG. With the exception of Bovine Anti-Goat IgG, many secondary antibodies such
as Anti-Bovine, Anti-Goat and Anti-Sheep will react strongly with bovine IgG. Therefore, use
However, binding of primaryantibodies to Fc receptors also may occur if they are whole of BSA or dry milk for blocking or diluting these antibodies may significantly increase
IgG antibodies, creating background regardless of the form of the secondaryantibody. To background and/or reduce antibody titre. For blocking, use normal serum (5% v/v) from the
block whole IgG primary and secondary antibodies from binding to Fc receptors, incubate host species of the labelled secondary antibody.
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Technical Information - Affinity-Purified Secondary Antibodies
Ordering e-mail: [email protected] Technical e-mail: [email protected]
Fab fragment(pages 46-52) antibodies are generated by papain digestion of whole IgG Step 4. Select the secondary antibody specificity under "Antibody Description".
antibodies to remove the entire Fc portion, including the hinge region (Figure 1). These The following explanations of terms may assist in selecting the most appropriate antibody
antibodies are monovalent, containing only a single antigen binding site. The molecular specificity.
weight of Fab fragments is about 50 kDa. They can be used to block endogenous
immunoglobulins on cells, tissues, or other surfaces and to block the exposed Note:Immunoglobulins from different species share similar structures, with similarities being
immunoglobulins in multiple labelling experiments using primary antibodies from the same related to closeness in phylogeny. Antibodies against immunoglobulins from one species
species. may cross-react with a number of other species unless they have been specifically adsorbed
against the cross-reacting species. Antibodies that have been adsorbed against other
In contrast, divalent (whole IgG or F(ab')2fragment) antibodies should not be used for species will contain "(min X...Sr Prot)" in the antibody description.
blocking since they have two binding sites. After blocking, some of the binding sites would
be available to capture the primary antibody introduced in a subsequent step, resulting in Anti-IgG (H+L)
higher background and/or coincidental labelling. These antibodies react with both the heavy and light chains of the IgG molecule, i.e. with
both the Fc and F(ab')2/Fab portions of IgG (Figure 1). Anti-IgG (H+L) antibodies also react
Step 2. Select the secondary antibody. with other immunoglobulin classes (IgM, IgA, IgD, IgE) and subclasses since they all share
The antibodies are listed alphabetically according to the host species of the primary the same light chains (either kappa or lambda). Anti-IgG (H+L) antibodies have broader
antibody. For example, if the primary antibody is made in mouse, go to the "Anti-Mouse" epitope recognition than anti-fragment specific antibodies. They are suggested for all general
section. immunodetection procedures.
Note:Both anti-Syrian and anti-Armenian hamster secondary antibodies are listed under Anti-IgG, Fc/Fc fragment specific
"Anti-Hamster". It is important to know in which strain of hamster the primary antibody γ
These antibodies react with the Fc portion of the IgG heavy chain. They have been tested
was produced since cross-reaction between the strains is not complete.
by ELISA and/or adsorbed against Fab fragments. In some cases, they are additionally
Step 3. Select the host species of the secondary antibody. tested and/or adsorbed to minimise cross-reactivity to IgM and/or IgA. In such cases (anti-
Selection of the host species for a secondary antibody involves many considerations, human, anti-mouse and anti-rat), they are labelled "Anti-IgG, Fc".
including but not limited to: 1) Antibodies from some host species may not be adsorbed γ
against cross-reacting species of interest. Choose a host species with the required Caution:Anti-IgG, Fc fragment specific antibodies may not react equally with all
adsorptions. 2) Host species compatibility. Some host species may not be compatible monoclonal primary antibγodies. For an anti-mouse IgG, Fc fragment specific antibody with
with other species in multiple-labelling experiments. In general, all secondary antibodies balanced reactivity to four subclasses of IgG, select goγat anti-mouse IgG (subclasses
for multiple labelling should come from the same host species. 3) Binding to Protein A 1+2a+2b+3), Fc fragment specific (min X Hu, Bov, Rb Sr Prot).
and Protein G. Rabbit antibodies bind well to Protein A and Protein G, but goat and γ
donkey antibodies bind better to Protein G. 4) Personal preference or experience. In our Anti-Mouse IgG, Fc Subclass specific
γ
experience there appears to be no host species-specific difference in the quality of These antibodies react with the Fc portion of the heavy chain of individual subclasses of
secondary antibodies. mouse IgG. They have been tested by ELISA and adsorbed to minimise cross-reactivity to
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Technical Information - Affinity-Purified Secondary Antibodies
other subclasses, Fab fragments, IgM and a few other species of IgG. Anti-Mouse IgG, The following abbreviations are used in the parentheses:
Fc subclass specific antibodies react with individual subclasses of mouse IgG. They are min X= minimal cross-reaction Ar Hms= Armenian Hamster Rb= Rabbit
inteγnded for distinguishing between different subclasses of mouse IgG primary antibodies Bov= Bovine Sy Hms= Syrian Hamster Shp= Sheep
in multiple labelling experiments, or for IgG subclass determination. Ck= Chicken Hrs= Horse Sw= Swine
Gt= Goat Hu= Human Sr= Serum
Anti-IgG, F(ab')2fragment specific GP= Guinea Pig Ms= Mouse Prot= Protein
These antibodies react with the F(ab')2/Fab portion of IgG. They have been tested by ELISA
and/or adsorbed against Fc fragments. They are not specific for IgG since they react with ML (multiple labelling)
light chains and therefore also react with other immunoglobulin classes (IgA, IgM, IgD and Some antibodies are designated "ML" to emphasise their usefulness in multiple labelling in
IgE) and subclasses sharing the same light chains. addition to single labelling. For further information see: "Multiple Labelling (ML) Using
Labelled Secondary Antibodies"(page 7).
(min X ... Sr Prot)
Secondary antibodies against one species may cross-react with other species unless they Anti-Armenian Hamster IgG vs Anti-Syrian Hamster IgG
have been specifically adsorbed against the other species. Antibodies with "(min X ... Sr Most hamster monoclonalantibodies are derived from Armenian hamster spleen cell-mouse
Prot)" in the description have been tested and/or adsorbed against IgG and/or serum myeloma hybridomas. The IgG produced by these hybridomas is Armenian (not Syrian)
proteins of those species indicated in the parentheses. They are recommended when the hamster IgG. Most commercially available polyclonalanti-hamster IgG antibodies have been
presence of immunoglobulins from other species may lead to interfering cross-reactivities. anti-Syrian hamster IgG, which are not as effective as anti-Armenian hamster IgG in detecting
However, caution should be exercised when considering antibodies that have been Armenian hamster IgG monoclonal antibodies.
adsorbed against closely related species, since they have greatly reduced epitope
recognition and may recognise some monoclonals poorly. For example, only use anti- Caution:Anti-Armenian Hamster IgG (H+L) (min X Bov, Hu, Ms, Rb, RatSr Prot) may not
mouse IgG adsorbed against rat IgG to detect a mouse primary antibody in rat tissue recognise all Armenian hamster monoclonal antibodies, since it has been adsorbed against
which contains endogenous rat immunoglobulins, or in a multiple labelling application closely related species (in bold). Therefore, it is better to use an antibody adsorbed against
which includes a rat primary antibody. Use anti-mouse IgG notadsorbed against rat IgG fewer species, such as Anti-Armenian Hamster IgG (H+L) (min X Bov Sr Prot), except in
to detect a mouse primary antibody in the absence of rat immunoglobulins. Two other those cases where Armenian hamster monoclonals need to be detected in the presence of
examples of antibodies which have diminished epitope recognition after adsorption with mouse and/or rat immunoglobulins.
closely related species are Anti-Rat IgG (min X ... Mouse ... Sr Prot) and Anti-Armenian
Hamster IgG (min X ... Mouse, Rat ... Sr Prot). Refer to "ML (Multiple Labelling)" for further Step 5. Select the desired probe from those listed at the top of the product
information. tables.
For technical information about probes, see "Technical Information on Probes Conjugated
to Affinity-Purified Antibodies and to Other Proteins"(page 8).
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Technical Information - Affinity-Purified Secondary Antibodies
Ordering e-mail: [email protected] Technical e-mail: [email protected]
Step 6. Find the size and code number of the selected antibody by following Multiple Labelling (ML) Using Labelled Secondary
the row until it intersects the column of the desired probe. Antibodies
The top numbers in each cell refer to the unit size. The bottom nine-digit number is the
catalogue code number. Selection of antibodies for simultaneous detection of more than one antigen depends on at
least two important criteria:
Step 7. Complete product description for ordering purposes. 1. Availability of secondary antibodies that do not recognise (a) one another (are derived from
For a complete description of the product, please use the standard format to avoid mistakes the same host species), (b) other primary antibodies used in the assay system, (c)
when placing an order. For example, a product with the code number 115-096-072 should immunoglobulins from other species present in the assay system, or (d) endogenous
be described as in the key below. immunoglobulins present in the tissues or cells under investigation.
2. Use of probes (enzyme-reaction products, fluorophores, or electron-dense particles) that
are well resolved.
FITC-conjugated AffiniPure F(ab')2fragment Goat Anti-Mouse IgG, F(ab')2fragment specific (min X Hu, Bov, Hrs Sr Prot)
The affinity-purified antibodies marked "ML" (multiple labelling) have been specifically
A B C D E F G
prepared to meet these criteria. One of many possible multiple-labelling protocols using these
A. Description of the probe, if it is conjugated. If unconjugated, nothing is required here. reagents is shown in the following example.
B. AffiniPure is our trade name for antibodies which have been isolated from antisera
by immunoaffinity chromatography using antigens coupled to agarose beads.
C. Form of the antibody - whole IgG, F(ab')2fragment, or Fab fragment antibody. Mouse Tissue Antigen A Mouse Tissue Antigen B Mouse Tissue Antigen C
D. Name of the host species of the secondary antibody.
E. Name of the species with which the antibody reacts. Step 1: 5% N. Donkey Serum to Step 4: 5% N. Donkey Serum to Step 7: 5% N. Donkey Serum
block block (if needed) to block (if needed)
F. Description of the antibody specificity.
G. List of species against which the antibody has been adsorbed to minimise cross-reactivity. Step 2: Goat Anti-Antigen A Step 5: Rabbit Anti-Antigen B Step 8: Rat Anti-Antigen C
Step 3: Probe l-conjugated Step 6: Probe ll-conjugated Step 9: Probe lll-conjugated
Key to the format of product descriptions eg: FITC-conjugated AffiniPure F(ab')2fragment Donkey Anti-Goat IgG Donkey Anti-Rabbit IgG (H+L) Donkey Anti-Rat IgG
Goat Anti-Mouse IgG, F(ab')2fragment specific (min X Hu, Bov, Hrs, Sr Prot) (H+L) (min X Ck, GP, Sy Hms, (min X Bov, Ck, Gt, GP, Sy Hms, (H+L) (min X Bov, Ck, Gt, GP,
Hrs, Hu, Ms, Rb, RatSr Prot) Hrs, Hu, Ms, Rat, Shp Sr Prot) Sy Hms, Hrs, Hu, Ms, Rb,
Shp Sr Prot)
Note:Wash thoroughly after each step, including after blocking at step 1. With heavy or persistent background
further blocking may be required at Steps 4 and 7. Do not dilute any antibody with normal serum or mix antibodies
together to save time, which may result in immune complex formation which could increase background.
In this example, the secondary antibodies used in Steps 3, 6 and 9 do not recognise each other since they are all
made in donkey. They have been solid-phase adsorbed so that they do not recognise the other primary antibodies
used in Steps 2, 5 and 8. Also, they do not react with endogenous mouse Ig, which may be present in the mouse
tissue.
For a review of multi-colour immunofluorescence labelling with confocal microscopy see Brelje, Wessendorf and
Sorenson, "Multi-colour laser scanning confocal immunofluorescence microscopy: Practical application and
limitations." In Cell Biological Applications of Confocal Microscopy (Methods in Cell Biology. vol.38). Ed. B.
Matsumoto. Orlando, FL: Academic Press, Inc. 1993, pp. 98-181.
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Technical Information - Probes
Technical Information on Probes Conjugated to
Fluorophore Excitation Peak Emission Peak (nm)
Affinity-Purified Antibodies and to Other Proteins.
Fluorescent Dyes
DyLight 405 400 421
(for Phycoerythrin, PerCP and Allophycocyanin conjugates, see page 53).
Aminomethylcoumarin, AMCA 350 450
The selection of fluorophores depends on:
A. Instrument set-up. Examples include availability of light sources, filter sets and detection Cyanine, Cy2 492 510
systems. Alexa Fluor®488 493 519
Fluorescein, FITC/DTAF 492 520
B. Degree of colour separation desired for multiple labelling. For example, to achieve
good colour separation from Alexa Fluor®488, choose a shorter wavelength emitting Indocarbocyanine, Cy3 550 570
fluorophore such as DyLight 405 and/or longer wavelength-emitting fluorophores such R-Phycoerythrin R-PE many, 488 580
as Rhodamine Red-X, Alexa Fluor®594, or Alexa Fluor®647. Rhodamine Red-X, RRX 570 590
Alexa Fluor®594 591 614
C. Sensitivity required. For example, Alexa Fluor®488 is brighter than FITC.
Allophycocyanin APC many, 650 660
The following fifteen fluorescent dye conjugates (Figure 2, Table 1) are currently available Alexa Fluor®647 651 667
from Jackson ImmunoResearch. They cover the most commonly used excitation sources
Indodicarbocyanine, Cy5 650 670
and filter sets from blue to infrared emissions.
Peridinan-Chlorophyll-protein, PerCP many, 488 675
Alexa Fluor®Fluorescent Dyes Alexa Fluor®680 684 702
Alexa Fluor®fluorescent dyes are widely recognised as superior fluorescent dyes available Alexa Fluor®790 792 803
for conjugation. They are highly water soluble and remain fluorescent from pH 4 to
pH 10. The detection level of any fluorophore-antibody conjugate depends on brightness
and photostability of the dye; antibody activity, specificity and cross-reactivity; and the Table 1.Approximate peak wavelengths of excitation and emission for all fluorophore-conjugated, affinity-purified
secondary antibodies offered by Jackson ImmunoResearch in order by increasing peak of emission. Approximate values
optimal moles of dye per mole of antibody. These parameters have been researched for are given for purposes of comparing one fluorophore with another. Actual values may vary depending on the
spectrofluorometer used in each laboratory.
each dye conjugate to optimise the level of antibody detection and minimise background.
Cyanine dyes (Cy2, Cy3 and Cy5) brighter in the non-polar environment than in an aqueous medium, resulting in less
Among currently available fluorescent dyes, the cyanine dyes are better able to withstand acquisition time than other dyes in the confocal microscope. The advantage of using plastic
the harsh dehydration and embedding conditions required for mounting sections in non- mounting media is better long-term storage for re-examination and retrieval of data at a
polar plastic mounting media, such as DPX and Permount™. The cyanine dyes are later date. See pages 68-70 for a list of cyanine dye conjugates.
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Technical Information - Probes
Ordering e-mail: [email protected] Technical e-mail: [email protected]
Figure 2.Excitation (left) and emission (right) spectra of
all fluorophores offered by Jackson ImmunoResearch.
The conjugates in order of increasing wavelength of
fluorescence are DyLight 405 (violet), AMCA (dark blue),
Cy2 (light blue), Alexa Fluor®488 (blue-green), FITC
(hidden behind Alexa Fluor®488), Cy3 (yellow), R-PE
Excitation Fluorescence CCA(adnlyyead55rx k) aA,( glPeFrpxeleuairenoC nkFrP®),lu , o(5 ldraA9®ovl4eet7t xne(9adrde0 e)Fd ,r(, )lb u,d loRaAorcht®lktlooe)dp6.d ah)4,my 7cA ionl(epcexyu aar pnRFlieenlu-d oh(-priX®din d6ke,8(n o0d rbo a(etngthgereiedny))d),,
This figure illustrates the relative shape and position of
each fluorophore in the peak region of its excitation and
fluorescence emission following conjugation to antibodies.
Quantitative comparisons should not be made since peak
0 0
heights have been normalised. All spectra were obtained
200 300 400 500 600 700 800 900 350 450 550 650 750 850
Wavelength (nm) Wavelength (nm) with an M-Series spectrofluorometer system from Photon
Technology International, Inc.
Figure 2.
Figure 3.Excitation (left) and Emission (right) spectra of
Alexa Fluor® conjugated affinity-purified secondary
antibodies, streptavidin and purified proteins. The dyes are
Alexa Fluor® 488 (blue), Alexa Fluor® 594 (green),
Alexa Fluor® 647 (red), Alexa Fluor® 680 (pink) and
Alexa Fluor®790 (black).
Excitation Fluorescence fTaslhunhoodisur oflidfpglu huonorerroee tisl luccbsoeetnnr ajcutmeeg saa edttehme e iin ssr seitnilhoacentei v. p eep Qsaehukaaa kprn eetgih taiaeontingidv heopt ofs s citiotshimo aenvpx eoac firt ibaesetoaieocnnnhs
normalised. All spectra were obtained with an M-Series
spectrofluorometer system from Photon Technology
International, Inc.
0 0
350 450 550 650 750 850 450 500 550 600 650 700 750 800 850
Wavelength (nm) Wavelength (nm)
Figure 3.
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Technical Information - Probes
Figure 4.Excitation (left) and emission (right) spectra
of Cy2 (green), Cy3 (red) and Cy5 (purple).
Peak heights were normalised after the spectra were
obtained with an M-series spectrofluorometer system
from Photon Technology International, Inc.
Excitation Fluorescence
0 0
300 350 400 450 500 550 600 650 700 400 450 500 550 600 650 700 750
Wavelength (nm) Wavelength (nm)
Figure 4.
Fluorescent Dyes in Order of Increasing Emission Wavelength cytometry, because emission filters generally used in flow cytometers are not optimal for
A brief description of the characteristics of fluorophores found in this catalogue is found DyLight 405. DyLight 405 conjugates are the best choice for blue-fluorescing secondary
below. The fluorophores are listed in order of increasing emission wavelength (Figure 2 and antibodies in multi-colour labelling protocols. See tables of Whole IgG Affinity-Purified
Table 1). For Phycoerythrin, PerCP and Allophycocyanin see page 53. Antibodies (pages 14-35), Streptavidin (page 71) and Purified Proteins from Normal
Serums (pages 80-89) for all DyLight 405 conjugates.
DyLight 405-conjugated secondary antibodies are excited maximally at about 400 nm
and fluoresce with a peak at about 421 nm (Figure 2 and Table 1). They are very bright AMCA (Aminomethylcoumarin Acetate)conjugates absorb light maximally around 350
and photostable, but their optimal use is limited to confocal microscopes equipped with a nm and fluoresce maximally around 450 nm (Figure 2 and Table 1). For fluorescence
405 nm laser and a 420 nm emission filter. Under these conditions, it is possible to microscopy, AMCA can be excited with a mercury lamp and observed using a UV filter set.
perform effective 4-colour imaging with good colour separation, good photostability and Since blue fluorescence is not well detected by the human eye, AMCA-conjugated
high sensitivity. The combination of DyLight 405, Alexa Fluor®488, Rhodamine Red-X and secondary antibodies should be used only with the most abundant antigens in multiple-
Alexa Fluor®647 provides for maximum colour separation (Figure 5). Another 4-colour labelling experiments. Ways of improving the visibility of AMCA include dark adapting the
dye combination, which may be equally effective but has slightly less colour separation, is eyes, using fluorite instead of glass objectives, avoiding mounting media that absorb UV
DyLight 405, Alexa Fluor® 488, Cy3 and Alexa Fluor® 647. DyLight 405 is not light (such as plastic-based media) and capturing photographic images with blue-sensitive
recommended for use in epifluorescence microscopes, nor is it recommended for flow film or CCD cameras. AMCA fades rapidly in conventional epifluorescence and confocal
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