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ELEVEN
CHAPTER 11
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Data Table 11.2 Probes for Tubulin and Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
Product List 11.2 Probes for Tubulin and Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
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477
Rhodamine Red goat antirabbit IgG, Alexa Fluor 488 goat antimouse IgG and Hoechst 33258.
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Fluorescent Actin
Alexa Fluor Actin and Unlabeled Actin
Fluorescently labeled actin (Figure 11.1.1) is an important tool for investigating the structural dynamics of the cytoskeleton.13 We offer highly
purified actin from rabbit muscle (A12375), as well as fluorescent actin conjugates labeled with four of our brightest and most photostable dyes. The
green-fluorescent Alexa Fluor 488 actin conjugate (A12373) has excitation
and emission maxima similar to fluorescein actin, but it is brighter and
more photostable, and its spectra are much less pH dependent. The redorangefluorescent Alexa Fluor 568 (A12374, Figure 11.1.2), red-fluorescent Alexa Fluor 594 (A34050) and far-redfluorescent Alexa Fluor 647
(A34051) actin conjugates are more fluorescent than the spectrally similar
Lissamine rhodamine B, Texas Red and Cy5 conjugates, respectively.
Our fluorescent actin conjugates are prepared by reacting amine
residues of polymerized F-actin with the succinimidyl ester of the appropriate dye using a modification of the method described by Alberts
and co-workers.4 After labeling, the conjugates are subjected to depolymerization and subsequent polymerization to help ensure that the
actin conjugates are able to assemble properly. The labeled actin that
polymerizes is then separated from remaining monomeric actin by centrifugation, depolymerized and packaged in monomeric form.
Figure 11.1.1 Ribbon diagram of the structure of uncomplexed actin in the ADP state. The four
subdomains are represented in different colors, and ADP is bound at the center where the four subdomains meet. Four Ca2+ ions bound to the actin monomer are represented as gold spheres. In this
structure, tetramethylrhodamine-5-maleimide (T6027) has been used to covalently attach the dye
to a specific cysteine residue (Cys 374). Image provided by Roberto Dominguez, Boston Biomedical
Research Institute, Watertown, Massachusetts. Reprinted with permission from Science (2001)
293:708. Copyright 2001 American Association for the Advancement of Science.
Figure 11.1.3 HeLa cell labeled with CellLight Actin-RFP (C10583) and CellLight MAP4-GFP
(C10598) reagents and with Hoechst 33342 nucleic acid stain.
Figure 11.1.2 Chick embryo fibroblasts injected with the Alexa Fluor 568 conjugate of actin
from rabbit muscle (A12374). The cells were then fixed and permeabilized, and the filamentous actin was stained with coumarin phallacidin (C606). The double-exposure image was
acquired using longpass filter sets appropriate for rhodamine and DAPI. Image contributed
by Heiti Paves, Laboratory of Molecular Genetics, National Institute of Chemical Physics and
Biophysics, Estonia.
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479
The CellLight Null (control) reagent (C10615), a suspension of baculovirus particles lacking mammalian genetic elements, is designed for
use in parallel with our CellLight reagents (Table 11.1). For example,
microarray expression analysis on cells treated with the CellLight Null
(control) reagent can be used to assess down-regulation or up-regulation of host cell genes elicited by baculovirus infection.
RFP
Handbook
GFP
Ref (489/508 nm)* (555/584 nm)* Section
Actin
Human actin
C10582
C10583
11.1
Tubulin
Human tubulin
C10613
C10614
11.2
MAP4
MAP4
C10598
C10599
11.2
Cat. No.
Talin
C10611
C10612
11.2
F-ActinSelective Probes
Chromatin
Histone 2B (H2B)
C10594
C10595
12.5
A22281
Mitochondria
Leader sequence
of E1 pyruvate
dehydrogenase
C10600
C10601
12.2
C606
Lysosomes
C10596
Peroxisomes
Peroxisomal C-terminal
targeting sequence
C10604
Rab5a
C10586
C10587
12.3, 16.1
Synaptosomes Synaptophysin
10
C10609
C10610
16.1
Endoplasmic
reticulum
11
C10590
C10591
12.4
Target
Endosomes
ER signal sequence of
calreticulin and KDEL
(ER retention signal)
12.3
12.3
12
Nucleus
LSV40 nuclear
localization sequence
13
C10602
C10603
12.5
Plasma
membrane
Myristoylation/
palmitoylation
sequence from Lck
tyrosine kinase
14
C10607
C10608
14.4
Golgi
apparatus
Cytoplasm
No targeting sequence
C10592
C10597
B10383
C10593
12.4
14.7
*Approximate absorption (Abs) and fluorescence (Em) maxima, in nm; GFP (Green
Fluorescent Protein) and RFP (Red Fluorescent Protein, Nat Methods (2007) 4:555) can
be imaged using optical filters for fluorescein (FITC) and tetramethylrhodamine (TRITC)
dyes, respectively. Also available is CellLight Plasma Membrane-CFP (C10606), which
generates a cyan-autofluorescent protein fused to the plasma membrane targeting
sequence from Lck tyrosine kinase.
1. Curr Biol (1997) 7:176; 2. PLoS One (2009) 4:e8171; 3. J Cell Biol (1995) 130:639; 4. Plant
J (2003) 33:775; 5. Curr Biol (1998) 8:377; 6. J Biol Chem (2004) 279:13044; 7. J Cell Sci (2005)
118:5243; 8. J Cell Biol (1989) 108:1657; 9. J Biol Chem (2009) 284:29218; 10. J Neurosci (2006)
26:3604; 11. FEBS Lett (1997) 405:18; 12. J Cell Biol (1998) 143:1505; 13. Trends Biochem Sci
(1991) 16:478; 14. EMBO J (1997) 16:4983.
Ex/Em*
Approximate MW
346/446
1100
Coumarin phallacidin
355/443
1100
N354
NBD phallacidin
465/536
1040
A12379
495/517
1320
F432
Fluorescein phalloidin
496/516
1175
O7466
496/520
1180
B607
BODIPY FL phallacidin
505/512
1125
O7465
511/528
1280
A22282
528/555
1350
R415
Rhodamine phalloidin
540/565
1250
A22283
554/570
1800
A34055
555/565
1800
B3475
558/569
1115
A12380
578/600
1590
A12381
593/617
1620
T7471
591/608
1490
A22284
625/645
1900
A34054
633/648
1900
B12382
647/661
1200
A22287
649/666
1950
A22285
661/689
1750
A22286
677/699
1850
B7474
Biotin-XX phalloidin
NA
1300
P3457
Phalloidin
NA
790
G-ActinSelective Probes
D12371
495/519
>31,000
D12372
590/617
>31,000
*Excitation (Ex) and emission (Em) maxima, in nm. Spectra of phallotoxins are either in
aqueous buffer, pH 79 (denoted ) or in methanol. Spectra of DNase I conjugates are in
aqueous buffer, pH 78. NA = Not applicable.
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NOTE 11.1
technology has many advantages when compared with lipids and other
viral delivery methods:
High transduction efficiency across a broad range of cell types, including primary and stem cells
Minimal microscopically observable cytopathic effects
ff
ffects
Highly reproducible and titratable transient expression
Biosafety level 1 rating (baculovirus is not pathogenic to
vertebrates and does not replicate in mammalian cells)
Ability to simultaneously deliver multiple genes
Furthermore, it is possible to divide the BacMam-transduced, homogeneous cell population into aliquots that can be stored frozen for use at a
later time, approximating the consistency of a stable cell line in a transient
expression format. More information is available at www.invitrogen.com/
handbook/bacmam2.0.
1. Nat Biotechnol (2004) 22:1583; 2. Br J Pharmacol (2008) 153:544; 3. Drug Discov
Today (2007) 12:396; 4. Nat Biotechnol (2005) 23:567; 5. Adv Virus Res (2006) 68:255.
Promoter
YFP (Venus)
Baculovirus
mRNA
translated
YFP (Venus)
Endocytotic entry
mRNA
DNA
DNA moves
to nucleus
Venus gene
transcribed
Figure 1 Schematic representation of BacMam transgene delivery and expression as exemplified by Premo Halide Sensor (P10229).
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481
The fluorescent and biotinylated phallotoxin derivatives stain F-actin selectively at nanomolar concentrations and are readily water soluble, thus providing convenient labels for identifying and quantitating actin in tissue sections, cell cultures or cell-free preparations.711
F-actin in live neurons can be efficiently labeled using cationic liposomes containing fluorescent phallotoxins, such as BODIPY FL phallacidin 12 (B607). This procedure permits the labeling of entire cell cultures with minimum disruption. Because fluorescent phalloidin conjugates
are not permeant to most live cells, they can be used to detect cells that have compromised
membranes. However, it has been reported that unlabeled phalloidin, and potentially dyelabeled phalloidins, can penetrate the membranes of certain hypoxic cells.13 An extensive study
on visualizing the actin cytoskeleton with various fluorescent probes in cell preparations, as
well as in live cells, has been published.7
Labeled phallotoxins have similar affinity for both large and small filaments and bind in a
stoichiometric ratio of about one phallotoxin per actin subunit in both muscle and nonmuscle
cells; they reportedly do not bind to monomeric G-actin, unlike some antibodies against actin.9,14
Phallotoxins have further advantages over antibodies for actin labeling, in that 1) their binding
properties do not change appreciably with actin from different species, including plants and
animals; and 2) their nonspecific staining is negligible; thus, the contrast between stained and
unstained areas is high.
Phallotoxins shift actins monomer/polymer equilibrium toward the polymer, lowering
the critical concentration for polymerization as much as 30-fold.15,16 Furthermore, depolymerization of F-actin by cytochalasins, potassium iodide and elevated temperatures is
inhibited by phallotoxin binding. Because the phallotoxin derivatives are relatively small,
with approximate diameters of 1215 and molecular weights below 2000 daltons, a wide
variety of actin-binding proteinsincluding myosin, tropomyosin, troponin and DNase I
can still bind to actin after treatment with f luorescent phallotoxins. Even more significantly,
phallotoxin-labeled actin filaments retain certain functional characteristics; labeled glycerinated muscle fibers still contract, and labeled actin filaments still move on solid-phase
myosin substrates.1719
We have taken advantage of the outstanding fluorescence characteristics of our Alexa Fluor
dyes (Section 1.3) to create a series of Alexa Fluor dyelabeled phalloidins (Figure 11.1.4, Figure
11.1.5, Figure 11.1.6, Figure 11.1.7), which are widely used F-actin stains for many applications
Figure 11.1.6 Subcellular structures in fixed and permeabilized bovine pulmonary artery endothelial cells visualized
with several fluorescent dyes. Filamentous actin (F-actin) was
identified with Alexa Fluor 633 phalloidin (A22284), which
is pseudocolored magenta. Intracellular membranes were
stained with green-fluorescent DiOC6(3) (D273). Finally, nuclei were counterstained with blue-fluorescent DAPI (D1306,
D3571, D21490). The image was acquired using filters appropriate for fluorescein and DAPI and a special filter (courtesy of
Omega Optical) for the Alexa Fluor 633 dye, consisting of a
narrow band exciter (630DF10), dichroic (640DRLP) and emitter (660DF10).
Figure 11.1.7 FluoCells prepared slide #4 (F24631) contains a section of mouse intestine stained with a combination of fluorescent stains. Alexa Fluor 350 wheat germ agglutinin (W11263) is a blue-fluorescent lectin that was used
to stain the mucus of goblet cells. The filamentous actin
prevalent in the brush border was stained with red-orange
fluorescent Alexa Fluor 568 phalloidin (A12380). Finally, the
nuclei were stained with SYTOX Green nucleic acid stain
(S7020). This image is a composite of three digitized images
obtained with filter sets appropriate for fluorescein, DAPI
and tetramethylrhodamine.
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across the full spectral range. The Alexa Fluor phalloidin conjugates
(Figure 11.1.8) provide researchers with fluorescent probes that are
superior in brightness and photostability to other spectrally similar
conjugates tested (Figure 11.1.9). For improved fluorescence detection
of F-actin in fixed and permeabilized cells, we encourage researchers
to try these fluorescent phalloidins in their actin-labeling protocols. A
series of videos showing Alexa Fluor 488 phalloidinstained actin 20
is available at the Journal of Cell Biology web site (www.jcb.org/cgi/
content/full/150/2/361/DC1).
SO3
H2N
SO3
NH2
C O
OH
O
CH3CH
NH
O C
HO
NH
CH3CCH2NH C
CH2
CH C NH CH C O
H2C
NH
CHCH3
H2C
N
H
N C CH NH C CH NH C O
O
O CHCH3
OH
Figure 11.1.10 Simultaneous visualization of F- and G-actin in a bovine pulmonary artery endothelial cell (BPAEC) using F-actinspecific Oregon Green 488 phalloidin (O7466) and G-actin
specific Texas Red deoxyribonuclease I. The G-actin appears as diffuse red fluorescence that
is more intense in the nuclear region where the cell thickness is greater and stress fibers are less
dense. The image was obtained by taking multiple exposures through bandpass optical filter sets
appropriate for fluorescein and the Texas Red dye.
Fluorescence (% of initial)
100
80
60
Oregon Green 514
40
20
Fluorescein
0
20
40
60
80
100
Time (seconds)
Figure 11.1.9 Comparison of the photobleaching rates of the Alexa Fluor 488 and Alexa Fluor 546 dyes and the well-known
fluorescein and Cy3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with
(top series) Alexa Fluor 488 phalloidin (A12379) and mouse monoclonal anti-tubulin antibody (A11126) in combination
with Alexa Fluor 546 goat antimouse IgG antibody (A11003) or (bottom series) fluorescein phalloidin (F432) and the antitubulin antibody in combination with a commercially available Cy3 goat antimouse IgG antibody. The pseudocolored images were taken at 30-second intervals (0, 30, 90 and 210 seconds of exposure). The images were acquired with bandpass filter
sets appropriate for fluorescein and rhodamine.
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BODIPY Phallotoxins
Figure 11.1.12 Permeabilized bovine pulmonary artery endothelial cells stained with SYTOX Green nucleic acid stain
(S7020) to label the nuclei and with BODIPY TR-X phallacidin (B7464) to label the F-actin. The image was acquired by
taking sequential exposures through bandpass optical filter
sets appropriate for fluorescein and the Texas Red dye.
BODIPY phallotoxin conjugates (B607, B3475, B12382; Figure 11.1.12, Figure 11.1.13) have
some important advantages over the conventional NBD, fluorescein and rhodamine phallotoxins. BODIPY dyes are more photostable than these traditional fluorophores 21 and have narrower
emission bandwidths (Section 1.4), making them especially useful for double- and triple-labeling
experiments. BODIPY FL phallacidin (B607), which reportedly gives a signal superior to that
of fluorescein phalloidin,22 has been used for quantitating F-actin and determining its distribution in cells.23,24
The BODIPY FL and BODIPY 558/568 phallotoxins (B607, B3475) exhibit excitation
and emission spectra similar to those of fluorescein and rhodamine B, respectively, and can
be used with standard optical filter sets. BODIPY 650/665 phalloidin (B12382) is the longestwavelength BODIPY phallotoxin conjugate available, increasing the options for multicolor
analysis. BODIPY 650/665 phalloidin, Alexa Fluor 647 phalloidin (A22287) and Alexa Fluor
660 phalloidin (A22285) are among the few probes available that can be excited by the 647 nm
spectral line of the Ar-Kr laser.
Rhodamine phalloidin (R415, Figure 11.1.14) has been the standard for red-fluorescent phallotoxins.2527 Rhodamine phalloidin is excited efficiently by the mercury-arc lamp in most fluorescence microscopes. However, our Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594 and
Texas Red-X phalloidins 28 (A22283, A12380, A12381, T7471; Figure 11.1.15, Figure 11.1.16) will
be welcome replacements for rhodamine phalloidin in many multicolor applications because
their emission spectra are better separated from those of the green-fluorescent Alexa Fluor 488,
Oregon Green and fluorescein dyes.
Bovine pancreatic deoxyribonuclease (DNase I, ~31,000 daltons) binds much more strongly
to monomeric G-actin than to filamentous F-actin, with binding constants of 5 108 M1 and
1.2 104 M1, respectively.3235 Because of this strong, selective binding to G-actin, fluorescent
DNase I conjugates have proven very useful for detecting and quantitating the proportion of
unpolymerized actin in a cell. We have triple-labeled endothelial cells with fluorescein DNase
I, BODIPY 581/591 phalloidin and a monoclonal anti-actin antibody detected with a Cascade
Blue dyelabeled secondary antibody 36 (C962, Section 7.2). We found that the monoclonal antibody, which binds to both G-actin and F-actin, colocalized with the DNase I and phalloidin conjugates, suggesting that these three probes recognize unique binding sites on the actin
molecule. Researchers can choose DNase I conjugates labeled with either the green-fluorescent
Alexa Fluor 488 (D12371) or red-fluorescent Alexa Fluor 594 (D12372) dyes, depending on their
multicolor application and their detection instrumentation (Table 11.2).
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Alexa Fluor 488 and Alexa Fluor 594 DNase I conjugates have been used in combination
with fluorescently labeled phallotoxins to simultaneously visualize G-actin pools and filamentous F-actin 37,38 and to study the disruption of microfilament organization in live nonmuscle
cells.39 Rhodamine phalloidin (R415) has been used in conjunction with Oregon Green 488
DNase I to determine the F-actin:G-actin ratio in Dictyostelium using confocal laser-scanning
microscopy.40 A mouse fibroblast labeled with both Texas Red DNase I and Oregon Green 488
phalloidin (O7466) permitted visualization of the G-actin and the complex network of F-actin
throughout the cytoplasm, as well as at the cell periphery (Figure 11.1.10). The influence of cytochalasins on actin structure in monocytes has been quantitated by flow cytometry using Texas
Red DNase I and BODIPY FL phallacidin (B607) to stain the G-actin and F-actin pools, respectively.41 Fluorescent DNase I has also been used as a model system to study the interactions
of nucleotides, cations and cytochalasin D with monomeric actin.42
Quantitative assays for F-actin have employed fluorescein phalloidin,43,44 rhodamine phalloidin,45 BODIPY FL phallacidin 24 and NBD phallacidin.46 An F-actin assay based on fluorescein phalloidin was used to demonstrate the loss of F-actin from cells during apoptosis.47 The addition of propidium iodide (P1304MP, P3566, P21493; Section 8.1) to the cell suspensions enabled
these researchers to estimate the cell-cycle distributions of both the apoptotic and nonapoptotic
cell populations. The change in F-actin content in proliferating adherent cells has been quantitated using the ratio of rhodamine phalloidin fluorescence to ethidium bromide fluorescence.48
The spectral separation of the signals in this assay may be improved by using a green-fluorescent
stain for F-actin and a high-affinity red-fluorescent nucleic acid stain, such as the combination of
Alexa Fluor 488 phalloidin (A12379) and ethidium homodimer-1 (E1169, Section 8.1).
The fluorescence of actin monomers labeled with pyrene iodoacetamide (P29) has been demonstrated to change upon polymerization, making this probe an excellent tool for following the
kinetics of actin polymerization and the effects of actin-binding proteins on polymerization.4951
Figure 11.1.16 Confocal micrograph of the cytoskeleton of a mixed population of granule neurons and glial
cells. The F-actin was stained with red-fluorescent Texas
Red-X phalloidin (T7471). The microtubules were detected with a mouse monoclonal anti-tubulin primary
antibody and subsequently visualized with the green-fluorescent Alexa Fluor 488 goat antimouse IgG antibody
(A11001). The image was contributed by Jonathan Zmuda,
Immunomatrix, Inc.
Figure 11.1.17 Bovine pulmonary artery endothelial cells were labeled with fluorescein phalloidin (F432), which labels filamentous actin, and placed under constant illumination on the microscope with a FITC filter set using a 60 objective. Images
were acquired at one-second intervals for 30 seconds. Under these illumination conditions, fluorescein photobleached to
about 12% of its initial value in 30 seconds in PBS (left), but stayed at the initial value under the same illumination conditions
when mounted using the reagents in the ProLong Antifade Kit (right, P7481).
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TheMolecular
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A Guide
to Fluorescent
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Labeling
Technologies
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We offer jasplakinolide (J7473, Figure 11.1.18), a macrocyclic peptide isolated from the
marine sponge Jaspis johnstoni.5254 Jasplakinolide is a potent inducer of actin polymerization
in vitro by stimulating actin filament nucleation 55,56 and competes with phalloidin for actin
binding 57 (Kd = 15 nM). Moreover, unlike other known actin stabilizers such as phalloidins and
virotoxins, jasplakinolide appears to be somewhat cell permeant and therefore can potentially
be used to manipulate actin polymerization in live cells. This peptide, which also exhibits fungicidal, insecticidal and antiproliferative activity, 53,5860 is particularly useful for investigating
cell processes mediated by actin polymerization and depolymerization, including cell adhesion,
locomotion, endocytosis and vesicle sorting and release. Jasplakinolide has been reported to
enhance apoptosis induced by cytokine deprivation.61
Latrunculins are powerful disruptors of microfilament organization. Isolated from a Red Sea
sponge, these G-actin binding compounds inhibit fertilization and early embryological development,62 alter the shape of cells 63,64 and inhibit receptor-mediated endocytosis.65 Latrunculin
A 61,63,66 (L12370, Figure 11.1.19) binds to monomeric G-actin in a 1:1 ratio at submicromolar concentrations (Howard Petty, Wayne State University, personal communication) and is frequently
used to establish the effects of F-actin disassembly on particular physiological functions such as
ion transport 67 and protein localization.68 The activity of latrunculin B (L22290) mimics that of
latrunculin A in most applications.63,65,6971
Figure 11.1.19 Latrunculin A (L12370).
REFERENCES
1. J Cell Biol (2009) 185:323; 2. J Am Chem Soc (2008) 130:16840; 3. Biophys J (2007) 92:1081; 4. Development (1988)
103:675; 5. Mol Biotechnol (2002) 21:241; 6. Proc Natl Acad Sci U S A (1974) 71:2803; 7. Microsc Res Tech (1999)
47:3; 8. Biophys J (1998) 74:2451; 9. Biophys J (2005) 88:2727; 10. Methods Enzymol (1991) 194:729; 11. J Muscle Res
Cell Motil (1988) 9:370; 12. Neurosci Lett (1996) 207:17; 13. J Lab Clin Med (1994) 123:357; 14. Biochemistry (1994)
33:14387; 15. Eur J Biochem (1987) 165:125; 16. J Cell Biol (1987) 105:1473; 17. J Cell Biol (1991) 115:67; 18. Nature
(1987) 326:805; 19. Proc Natl Acad Sci U S A (1986) 83:6272; 20. J Cell Biol (2000) 150:361; 21. J Cell Biol (1991)
114:1179; 22. J Cell Biol (1994) 127:1637; 23. J Cell Biol (1992) 116:197; 24. Histochem J (1990) 22:624; 25. Biochemistry
(2008) 47:6460; 26. BMC Cell Biol (2007) 8:43; 27. Biotechniques (2006) 40:745; 28. J Histochem Cytochem (2001)
49:1351; 29. J Muscle Res Cell Motil (1993) 14:594; 30. J Cell Biol (1995) 130:591; 31. Biol Chem (1998) 273:17128;
32. Anal Biochem (1983) 135:22; 33. Exp Cell Res (1983) 147:240; 34. Eur J Biochem (1980) 104:367; 35. J Biol Chem
(1980) 255:5668; 36. J Histochem Cytochem (1994) 42:345; 37. Stem Cells (2005) 23:507; 38. Am J Physiol Heart Circ
Physiol (2005) 288:H660; 39. Proc Natl Acad Sci U S A (1990) 87:5474; 40. J Cell Biol (1998) 142:1325; 41. J Biol Chem
(1994) 269:3159; 42. Eur J Biochem (1989) 182:267; 43. Proc Natl Acad Sci U S A (1980) 77:6624; 44. J Cell Sci (1991)
100:187; 45. J Cell Biol (1995) 130:613; 46. J Cell Biol (1984) 98:1265; 47. Cytometry (1995) 20:162; 48. J Cell Biol
(1995) 129:1589; 49. Curr Biol (2006) 16:1924; 50. J Biol Chem (2008) 283:7135; 51. Biophys J (2007) 92:2162; 52. J Cell
Biol (1997) 137:399; 53. J Am Chem Soc (1986) 108:3123; 54. Tetrahedron Lett (1986) 27:2797; 55. Methods Mol Biol
(2001) 161:109; 56. J Biol Chem (2000) 275:5163; 57. J Biol Chem (1994) 269:14869; 58. J Natl Cancer Inst (1995) 87:46;
59. Cancer Chemother Pharmacol (1992) 30:401; 60. Antimicrob Agents Chemother (1988) 32:1154; 61. J Biol Chem
(1999) 274:4259; 62. Science (1983) 219:493; 63. J Biol Chem (2000) 275:28120; 64. FEBS Lett (1987) 213:316; 65. Exp
Cell Res (1986) 166:191; 66. Cell Motil Cytoskeleton (1989) 13:127; 67. J Biol Chem (1997) 272:20332; 68. Am J Physiol
(1997) 272:C254; 69. J Biol Chem (2001) 276:23056; 70. J Cell Sci (2001) 114:1025; 71. Cell Motil Cytoskeleton (2001)
48:96; 72. Anal Biochem (1992) 200:199; 73. J Biol Chem (1994) 269:32916.
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none
1, 2
B7474
~1300
F
MeOH, H2O
B12382
~1200
F,L
MeOH
647
102,000
661
MeOH
1, 3, 5
355
16,000
443
MeOH
1, 2, 3
C606
~1100
F,L
MeOH, H2O
496
84,000
516
pH 8
1, 2, 3
F432
~1175
F,L
MeOH, H2O
J7473
709.68
F,D
MeOH
278
8000
none
MeOH
L12370
421.55
F,D
DMSO
<300
none
L22290
395.51
F,D
DMSO
<300
none
465
24,000
536
MeOH
1, 2, 3
N354
~1040
F,L
MeOH, H2O
511
85,000
528
pH 9
1, 2, 3
O7465
~1280
F,L
MeOH, H2O
496
86,000
520
pH 9
1, 2, 3
O7466
~1180
F,L
MeOH, H2O
P29
385.20
F,D,L
DMF, DMSO
339
26,000
384
MeOH
6, 7
<300
see Notes
2, 8
P3457
~790
F
MeOH, H2O
542
85,000
565
MeOH
1, 2, 3, 9
R415
~1250
F,L
MeOH, H2O
583
95,000
603
MeOH
1, 2, 3, 9
T7471
~1490
F,L
MeOH, H2O
For definitions of the contents of this data table, see Using The Molecular Probes Handbook in the introductory pages.
Notes
1. -Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.
2. Although this phallotoxin is water-soluble, storage in water is not recommended, particularly in dilute solution.
3. The value of EC listed for this phallotoxin conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC due to
dyepeptide interactions.
4. In aqueous solutions (pH 7.0), Abs/Em = 625/645 nm for A22284, 633/648 nm for A34054, 649/666 nm for A22287, 661/689 nm for A22285 and 677/699 nm for A22286.
5. B7464 and B12382 are not directly soluble in H2O. Aqueous dispersions can be prepared by dilution of a stock solution in MeOH.
6. Spectral data of the 2-mercaptoethanol adduct.
7. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
8. This bicyclic peptide is very weakly fluorescent in aqueous solution (Em ~380 nm). (Biochim Biophys Acta (1983) 760:411)
9. In aqueous solutions (pH 7.0), Abs/Em = 554/573 nm for R415 and 591/608 nm for T7471.
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Product
A12375
A12373
A12374
A34050
A34051
A22281
A12379
A22282
A22283
A34055
A12380
A12381
A22284
A34054
A22287
A22285
A22286
B7474
B3475
B12382
B607
C10582
C10583
C10615
C606
D12371
D12372
F432
J7473
L12370
L22290
N354
O7466
O7465
P3457
P29
R415
T7471
Quantity
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1 mg
200 g
200 g
200 g
200 g
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
300 U
50 U
300 U
300 U
300 U
1 mL
1 mL
1 mL
300 U
5 mg
5 mg
300 U
100 g
100 g
100 g
300 U
300 U
300 U
1 mg
100 mg
300 U
300 U
TubulinTracker Green reagent (T34075) provides green-fluorescent staining of polymerized tubulin in live cells.911 Also known as
Oregon Green 488 paclitaxel bis-acetate (a bi-acetylated version of
Oregon Green 488 paclitaxel (P22310), see below), TubulinTracker
Green reagent is an uncharged, nonfluorescent compound (Figure
11.2.1) that easily passes through the plasma membrane of live cells. Once
inside the cell, the lipophilic blocking group is cleaved by nonspecific
esterases, resulting in a green-fluorescent, charged paclitaxel.
TubulinTracker Green reagent is provided as a set of two components: lyophilized TubulinTracker Green reagent and a 20% Pluronic
F-127 solution in dimethylsulfoxide (DMSO), a solubilizing agent for
making stock solutions and facilitating cell loading. Please note that
because paclitaxel binds polymerized tubulin, TubulinTracker Green
reagent will inhibit cell division and possibly other functions utilizing
polymerized tubulin in live cells.
In addition to unlabeled paclitaxel and TubulinTracker Green reagent, we provide three fluorescent derivatives of paclitaxel: Oregon
Green 488 paclitaxel (Flutax-2, P22310), BODIPY FL paclitaxel
(P7500) and BODIPY 564/570 paclitaxel (P7501). These fluorescent
paclitaxel derivatives are promising tools for imaging microtubule
formation and motility. Their fluorescent attributes should also make
these conjugates useful reagents for screening compounds that affect
microtubule assembly.
Oregon Green 488 paclitaxel 1216 is an important probe for labeling tubulin filaments in live cells. The fluorescent label on this probe
is attached by derivatizing the 7-hydroxy group of native paclitaxel
(Figure 11.2.2), a strategy that permits selective binding of the probe to
microtubules with high affinity at 37C 16 (Kd ~10 7 M). Oregon Green
488 paclitaxel has been utilized in a high-throughput fluorescence polarizationbased assay to screen for paclitaxel biomimetics.14 We have
successfully used Oregon Green 488 paclitaxel to label microtubules
CH3CO
OCCH3
F
O
C O
CH3
C O
H3C
CH3
CH3
CH3
C NHCHCH C O
OH
HO
O O
O C C O
CH3
C O
O C CH2CH2NH
Figure 11.2.1 TubulinTracker Green (Oregon Green 488 Taxol, bis-acetate; T34075).
Figure 11.2.2 Paclitaxel, Oregon Green 488 conjugate (Oregon Green 488 Taxol,
Flutax-2; P22310).
The
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of live HeLa (Figure 11.2.3), NIH 3T3, A-10 and BC3H1 cells. Xenopus
laevis17 and bovine brain 18 microtubules have also been stained with
Oregon Green 488 paclitaxel.
In the BODIPY FL and BODIPY 564/570 paclitaxel derivatives,
the N-benzoyl substituent of the 3-phenylisoserine portion of native
paclitaxel is replaced by a BODIPY propionyl substituent (Figure
11.2.4). As an alternative to chemically modifying tubulin with a reactive fluorophore, a published method describes the use of these
BODIPY paclitaxel derivatives to generate fluorescent microtubules
that are stable at room temperature for one week or longer.19 In contrast
to the Oregon Green 488 derivative, the BODIPY FL and BODIPY
564/570 paclitaxel derivatives do not appear to be suitable for labeling
intracellular tubulin in most cases.
Tubulin-Selective Probes
Figure 11.2.3 Microtubules were assembled, stabilized and visualized with the aid of greenfluorescent Oregon Green 488 paclitaxel (P22310). Viable HeLa cells were incubated with the
conjugate for 1 hour, followed by several washes with phosphate-buffered saline containing
2% bovine serum albumin. The image was acquired using a confocal laser-scanning microscope and a filter set appropriate for fluorescein.
GFPtubulin fusions are well-established probes for imaging cytokinesis and other dynamic rearrangements of microtubules in live
cells.20 CellLight Tubulin-GFP and CellLight Tubulin-RFP expression vectors (C10613, C10614; Table 11.1) generate autofluorescent proteins fused to the N-terminus of human -tubulin and incorporate all
the generic advantages of BacMam 2.0 delivery technology (BacMam
Gene Delivery and Expression TechnologyNote 11.1).
In context-specific instances where GFPtubulin fusion protein
incorporation into microtubules is inefficient, CellLight expression
vectors encoding GFP (C10598; Figure 11.2.5) or RFP (C10599) fused
to the N-terminus of the mammalian microtubule-associated protein
MAP4 provide a second option for microtubule visualization. However,
because MAP4 stabilizes polymerized tubulin, CellLight Tubulin-GFP
and CellLight Tubulin-RFP are generally preferable for molecular-level
investigations of microtubule dynamic instability.
Figure 11.2.6 Microtubules of bovine pulmonary artery endothelial cells tagged with mouse monoclonal anti-tubulin antibody (A11126) and subsequently probed with: Alexa Fluor 488 goat antimouse IgG antibody (A11001, left), Alexa Fluor
546 goat antimouse IgG antibody (A11003, middle) or Alexa Fluor 594 goat antimouse IgG antibody (A11005, right). These
images were acquired using a fluorescein bandpass optical filter set, a rhodamine bandpass optical filter set and a Texas Red
bandpass optical filter set, respectively.
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When used in conjunction with an antimouse IgG secondary immunoreagent (Section 7.2, Table 7.1), our anti-tubulin monoclonal
antibody (A11126) enables researchers to visualize microtubules in
fixed cells (Figure 11.2.6, Figure 11.2.7, Figure 11.2.8, Figure 11.2.9)
and in fixed or frozen tissue sections from various species. This mouse
monoclonal antibody, which recognizes amino acid residues 6997 of
the N-terminal structural domain, is also useful for detecting tubulin
by ELISA or western blotting, for screening expression libraries and as
a probe for the N-terminal domain of -tubulin.
The anti-tubulin monoclonal antibody is available either unlabeled (A11126) or as a biotin-XX conjugate (A21371). For detecting the
biotinylated antibody, we carry a wide variety of fluorophore- and enzyme-labeled avidin, streptavidin and NeutrAvidin biotin-binding protein conjugates and NANOGOLD and Alexa Fluor FluoroNanogold
streptavidin (Section 7.6, Table 7.9).
We have extensively utilized the mouse IgG1 monoclonal antitubulin antibody during development and evaluation of our Zenon
technology (Section 7.3, Table 7.7), which allows labeling of submicrogram quantities of primary antibodies in minutes (Figure 11.2.10,
Figure 11.2.11). A comprehensive listing of our primary antibodies for cytoskeletal proteins can be found at www.invitrogen.com/
handbook/antibodies.
Figure 11.2.7 Microtubules of fixed bovine pulmonary artery endothelial cells were labeled
with our mouse monoclonal anti-tubulin antibody (A11126), detected with the biotin-XXconjugated F(ab)2 fragment of goat antimouse IgG antibody (B11027) and visualized with Alexa
Fluor 488 streptavidin (S11223). The actin filaments were then labeled with orange-fluorescent
Alexa Fluor 568 phalloidin (A12380), and the cell was counterstained with blue-fluorescent
Hoechst 33342 (H1399, H3570, H21492) to image the DNA, and red-fluorescent propidium iodide
(P1304MP, P3566, P21493) to image the nucleolar RNA. The multiple-exposure image was acquired
using bandpass filters appropriate for the Texas Red dye, fluorescein and DAPI.
Figure 11.2.8 Bovine pulmonary artery endothelial cells were labeled with Alexa Fluor
488 phalloidin (A12379) to stain F-actin and our mouse monoclonal anti-tubulin antibody
(A11126) in combination with Alexa Fluor 594 dyeconjugated F(ab)2 fragment of goat
antimouse IgG antibody (A11020) to stain microtubules. The multiple-exposure image was
acquired using bandpass filter sets appropriate for Texas Red dye and fluorescein.
Figure 11.2.10 Fixed and permeabilized bovine pulmonary artery endothelial cells stained with Alexa Fluor 350
phalloidin (A22281), an anti-tubulin antibody (A11126)
and the anticdc6 peptide antibody (A21286). The antitubulin antibody was labeled with the Zenon Alexa Fluor
568 Mouse IgG1 Labeling Kit (Z25006) and the anticdc6
peptide antibody was labeled with the Zenon Alexa Fluor
488 Mouse IgG1 Labeling Kit (Z25002).
The
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491
BODIPY FL Vinblastine
Talin is a cytoskeletal protein that is concentrated in focal adhesions, linking integrins to the actin cytoskeleton either directly
or indirectly by interacting with vinculin and -actinin. CellLight
Talin-GFP and CellLight Talin-RFP expression vectors (C10611,
C10612; Table 11.1; Figure 11.2.13) generate autof luorescent proteins fused to the C-terminal actin-binding domain of human talin
and incorporate all the generic advantages of BacMam 2.0 delivery
technology (BacMam Gene Delivery and Expression Technology
Note 11.1). These CellLight reagents have potential applications
in image-based high-content screening (HCS) assays of integrinmediated cell adhesion, as well as for general-purpose labeling of
cytoskeletal actin in live cells.
Figure 11.2.13 HeLa cell labeled with CellLight Talin-GFP (C10611) and CellLight Actin-RFP
(C10583) reagents.
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Anti-Desmin Antibody
Figure 11.2.14 Rat brain cryosections were labeled with the red-fluorescent Alexa Fluor 594
conjugate of antiglial fibrillary acidic protein antibody (A21295). Nuclei were counterstained
with TOTO-3 iodide (T3604, pseudocolored blue).
Anti-Synapsin I Antibody
Figure 11.2.15 The intermediate filaments in bovine pulmonary artery endothelial cells, localized using our anti-desmin antibody (A21283), which was visualized with the Alexa Fluor 647
goat antimouse IgG antibody (A21235). Endogenous biotin in the mitochondria was labeled
with Alexa Fluor 546 streptavidin (S11225) and DNA in the cell was stained with blue-fluorescent DAPI (D1306, D3571, D21490).
Figure 11.2.16 Peripheral neurons in mouse intestinal cryosections were labeled with rabbit
antisynapsin I antibody (A6442) and detected using Alexa Fluor 488 goat antirabbit IgG
antibody (A11008). This tissue was counterstained with DAPI (D1306, D3571, D21490).
The
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REFERENCES
1. BMC Cancer (2006) 6:86; 2. J Am Chem Soc (1971) 93:2325; 3. J Am Chem Soc
(1988) 110:5917; 4. Tetrahedron (1986) 42:4451; 5. J Biol Chem (1994) 269:23399;
6. J Cell Biol (1991) 112:1177; 7. Pharmacol Ther (1984) 25:83; 8. Cancer Treat Rep (1978)
62:1219; 9. PLoS Biol (2008) 6:e209; 10. J Neurosci (2008) 28:2601; 11. J Neurochem
(2007) 102:1009; 12. J Biol Chem (2003) 278:8407; 13. Biochemistry (2002) 41:12436;
14. Biochemistry (2001) 40:11975; 15. Cell Motil Cytoskeleton (2001) 49:1; 16. J Biol
Chem (2000) 275:26265; 17. J Cell Biol (2000) 148:883; 18. Chem Biol (2000) 7:275;
19. Biotechniques (1998) 25:188; 20. Mol Biotechnol (2002) 21:241; 21. Mol Pharmacol
(2002) 62:1238; 22. Cancer Res (2002) 62:6864; 23. Mol Biol Cell (1995) 6:1215; 24. Mol
Pharmacol (2002) 62:1; 25. FEBS Lett (1997) 416:251; 26. J Biol Chem (1996) 271:14707;
27. Eur J Biochem (1997) 244:664; 28. J Neurochem (1996) 67:1688; 29. Biochemistry
(1994) 33:12665; 30. Acta Histochem (1993) 94:54; 31. Arch Biochem Biophys
(1993) 303:159; 32. Eur J Biochem (1987) 165:613; 33. J Biol Chem (1985) 260:2819;
34. Anal Biochem (2003) 315:49; 35. J Biol Chem (1984) 259:14647; 36. Biochemistry
(1994) 33:11900; 37. Biochemistry (1994) 33:11891; 38. Biochemistry (1986) 25:3536;
39. Biochemistry (1998) 37:4687; 40. Biochemistry (1995) 34:13367; 41. Cell (1990) 62:579;
42. Biochemistry (1989) 28:6678; 43. Immunol Lett (1992) 33:285; 44. J Biochem (Tokyo)
(1991) 109:499; 45. J Biol Chem (1990) 265:14899; 46. Eur J Biochem (1992) 204:127;
47. Neurochem Res (2000) 25:1439; 48. Biochem Biophys Res Commun (1995) 208:910;
49. Biochim Biophys Acta (1996) 1313:268; 50. J Neurol Sci (1997) 151:41; 51. Proc Natl
Acad Sci U S A (1976) 73:4344; 52. J Cell Sci (1977) 23:243; 53. EMBO J (1982) 1:1649;
54. Acta Cytol (2000) 44:976; 55. Science (1984) 226:1209; 56. J Cell Biol (1983) 96:1337.
DATA TABLE 11.2 PROBES FOR TUBULIN AND OTHER CYTOSKELETAL PROTEINS
Cat. No.
MW
Storage
Soluble
Abs
EC
Em
Solvent
Notes
B153
672.85
L
pH >6
395
23,000
500
MeOH
1, 2
342
28,000
450
pH 7
3
D1306
350.25
L
H2O, DMF
342
28,000
450
pH 7
3
D3571
457.49
L
H2O, MeOH
D3923
249.31
L
DMF, DMSO
456
61,000
493
MeOH
4
342
28,000
450
pH 7
3, 5
D21490
350.25
L
H2O, DMF
N1142
318.37
L
DMF, DMSO
552
45,000
636
MeOH
6
P248
227.31
L
DMF, MeCN
363
19,000
497
MeOH
7
P3456
853.92
F,D
MeOH, DMSO
228
30,000
none
MeOH
P7500
1023.89
FF,D,L
DMSO
504
66,000
511
MeOH
P7501
1098.98
FF,D,L
DMSO
565
121,000
571
MeOH
P22310
1319.28
FF,D,L
DMSO, EtOH
494
80,000
522
pH 9
V12390
1043.02
F,D,L
DMSO, DMF
503
83,000
510
MeOH
For definitions of the contents of this data table, see Using The Molecular Probes Handbook in the introductory pages.
Notes
1. B153 is soluble in water at 0.11.0 mM after heating.
2. Bis-ANS (B153) bound to tubulin has Abs = 392 nm, Em = 490 nm and a fluorescence quantum yield of 0.23. (Biochemistry (1994) 33:11900)
3. DAPI undergoes an approximately 9-fold fluorescence enhancement on binding to polymerized tubulin. Abs = 345 nm, Em = 446 nm. (J Biol Chem (1985) 260:2819)
4. The absorption and fluorescence emission maxima of DCVJ (D3923) bound to tubulin are essentially the same as in methanol. (Biochemistry (1989) 28:6678)
5. This product is specified to equal or exceed 98% analytical purity by HPLC.
6. The fluorescence emission maximum of nile red (N1142) bound to tubulin is 623 nm. (J Biol Chem (1990) 265:14899)
7. The fluorescence emission maximum of prodan (P248) bound to tubulin is ~450 nm. (Eur J Biochem (1992) 204:127)
PRODUCT LIST 11.2 PROBES FOR TUBULIN AND OTHER CYTOSKELETAL PROTEINS
Cat. No.
Product
A21283
A21282
A21294
A21295
A6442
A11126
A21371
B153
C10598
C10599
C10611
C10612
C10613
C10614
D3923
D1306
D21490
D3571
N1142
P3456
P7501
P7500
P22310
P248
T34075
V12390
Quantity
The
MolecularProbes
Probes Handbook:
Handbook: AA Guide
Probesand
andLabeling
LabelingTechnologies
Technologies
The
Molecular
Guide to
to Fluorescent
Fluorescent Probes
494
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
the Appendix on
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
www.invitrogen.com/probes
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100 L
100 L
50 L
50 L
10 g
50 g
50 g
10 mg
1 mL
1 mL
1 mL
1 mL
1 mL
1 mL
25 mg
10 mg
10 mg
10 mg
25 mg
5 mg
10 g
10 g
100 g
100 mg
1 set
100 g