The Molecular Probes® Handbook A GUIDE TO FLUORESCENT … · 2020. 10. 31. · CHAPTER 1...

CHAPTER 11

Probes for Cytoskeletal Proteins

Molecular Probes™ HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes® HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

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ELE

VEN

CHAPTER 11

Probes for Cytoskeletal Proteins

11.1 Probes for Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Fluorescent Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Alexa Fluor® Actin and Unlabeled Actin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

GFP- and RFP-Labeled Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

CellLight® Null Control Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480

Phallotoxins for Labeling F-Actin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480

Properties of Phallotoxin Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482

Alexa Fluor® Phalloidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482

Oregon Green® Phalloidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

BODIPY® Phallotoxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

Rhodamine Phalloidin and Other Red-Fluorescent Phalloidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

Other Labeled Phallotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

DNase I Conjugates for Staining G-Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484

Probes for Actin Quantitation, Actin Polymerization and Actin-Binding Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

Assays for Quantitating F-Actin and G-Actin Polymerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

Jasplakinolide: A Cell-Permeant F-Actin Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

Latrunculin A and Latrunculin B: Cell-Permeant Actin Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

Assays for Actin-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486

Data Table 11.1 Probes for Actin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

Product List 11.1 Probes for Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488

11.2 Probes for Tubulin and Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

Paclitaxel Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

TubulinTracker™ Green Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

Fluorescent Paclitaxel Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

Tubulin-Selective Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

GFP- and RFP-Labeled Tubulin and MAP4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

Anti–α-Tubulin Monoclonal Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

BODIPY® FL Vinblastine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

Other Probes for Tubulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

Probes for Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

GFP- and RFP-Labeled Talin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

Anti–Glial Fibrillary Acidic Protein (GFAP) Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492

Anti-Desmin Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

Anti-Synapsin I Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

Data Table 11.2 Probes for Tubulin and Other Cytoskeletal Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

Product List 11.2 Probes for Tubulin and Other Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies

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 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.

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Chapter 11 — Probes for Cytoskeletal Proteins

Rhodamine Red™ goat anti–rabbit IgG, Alexa Fluor® 488 goat anti–mouse IgG and Hoechst 33258.


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Section 11.1 Probes for Actin

11.1 Probes for Actin

Figure 11.1.1 Ribbon diagram of the structure of uncomplexed actin in the ADP state. The four subdomains are represented in di�erent colors, and ADP is bound at the center where the four sub-domains 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 speci�c 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.

�e cytoskeleton is an essential component of a cell’s structure and one of the easiest to label with �uorescent reagents. �is section de-scribes Molecular Probes® labeling reagents for both monomeric actin (G-actin) and �lamentous actin (F-actin); reagents for staining tubulin and other cytoskeletal proteins are described in Section 11.2.

Fluorescent ActinAlexa Fluor® Actin and Unlabeled Actin

Fluorescently labeled actin (Figure 11.1.1) is an important tool for in-vestigating the structural dynamics of the cytoskeleton.1–3 We o�er highly puri�ed actin from rabbit muscle (A12375), as well as �uorescent actin con-jugates labeled with four of our brightest and most photostable dyes. �e green-�uorescent Alexa Fluor® 488 actin conjugate (A12373) has excitation and emission maxima similar to �uorescein actin, but it is brighter and more photostable, and its spectra are much less pH dependent. �e red-orange–�uorescent Alexa Fluor® 568 (A12374, Figure 11.1.2), red-�uores-cent Alexa Fluor® 594 (A34050) and far-red–�uorescent Alexa Fluor® 647 (A34051) actin conjugates are more �uorescent than the spectrally similar Lissamine rhodamine B, Texas Red® and Cy®5 conjugates, respectively.

Our �uorescent actin conjugates are prepared by reacting amine residues of polymerized F-actin with the succinimidyl ester of the ap-propriate dye using a modi�cation of the method described by Alberts and co-workers.4 A�er labeling, the conjugates are subjected to depo-lymerization and subsequent polymerization to help ensure that the actin conjugates are able to assemble properly. �e labeled actin that polymerizes is then separated from remaining monomeric actin by cen-trifugation, depolymerized and packaged in monomeric form.

GFP- and RFP-Labeled Actin�e requirement for intracellular delivery of Alexa Fluor® dye–

labeled actin conjugates by microinjection typically limits their applica-tions for live-cell imaging to experiments involving no more than a few (<10) cells. For applications such as high-content screening (HCS) assays requiring larger sample sizes, GFP–actin fusions are well-established probes for imaging cytoskeletal structure and dynamics.5 CellLight® Actin-GFP (C10582) and CellLight® Actin-RFP (C10583, Figure 11.1.3)

Figure 11.1.2 Chick embryo �broblasts injected with the Alexa Fluor® 568 conjugate of actin from rabbit muscle (A12374). The cells were then �xed and permeabilized, and the �lamen-tous actin was stained with coumarin phallacidin (C606). The double-exposure image was acquired using longpass �lter sets appropriate for rhodamine and DAPI. Image contributed by Heiti Paves, Laboratory of Molecular Genetics, National Institute of Chemical Physics and Biophysics, Estonia.

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.



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Table 11.1 CellLight® reagents and their targeting sequences.

Target Targeting Sequence RefGFP (489/508 nm)*

RFP (555/584 nm)*

Handbook Section

Actin Human actin 1 C10582 C10583 11.1

Tubulin Human tubulin 2 C10613 C10614 11.2

MAP4 MAP4 3 C10598 C10599 11.2

Talin Human talin 2341–2541 4 C10611 C10612 11.2

Chromatin Histone 2B (H2B) 5 C10594 C10595 12.5

Mitochondria Leader sequence of E1α pyruvate dehydrogenase

6 C10600 C10601 12.2

Lysosomes Lamp1 (lysosomal-associated membrane protein 1)

7 C10596 C10597 12.3

Peroxisomes Peroxisomal C-terminal targeting sequence

8 C10604 12.3

Endosomes Rab5a 9 C10586 C10587 12.3, 16.1

Synaptosomes Synaptophysin 10 C10609 C10610 16.1

Endoplasmic reticulum

ER signal sequence of calreticulin and KDEL (ER retention signal)

11 C10590 C10591 12.4

Golgi apparatus

Human golgi-resident enzyme N-acetylgalactos-aminyltransferase 2

12 C10592 C10593 12.4

Nucleus LSV40 nuclear localization sequence

13 C10602 C10603 12.5

Plasma membrane

Myristoylation/palmitoylation sequence from Lck tyrosine kinase

14 C10607 † C10608 † 14.4

Cytoplasm No targeting sequence B10383 14.7

* Approximate absorption (Abs) and �uorescence (Em) maxima, in nm; GFP (Green Fluorescent Protein) and RFP (Red Fluorescent Protein, Nat Methods (2007) 4:555) can be imaged using optical �lters for �uorescein (FITC) and tetramethylrhodamine (TRITC) dyes, respectively. † Also available is CellLight® Plasma Membrane-CFP (C10606), which generates a cyan-auto�uorescent 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.

expression vectors (Table 11.1) generate auto�uorescent proteins fused to the N-terminus of human β-actin and incorporate all the generic advan-tages of BacMam 2.0 delivery technology (BacMam Gene Delivery and Expression Technology—Note 11.1). In particular, the viral dose can be readily adjusted to modulate expression levels if GFP- or RFP-dependent perturbation of cellular structural or functional properties is a concern.

CellLight® Null Control Reagent�e CellLight® Null (control) reagent (C10615), a suspension of bac-

ulovirus 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-regula-tion of host cell genes elicited by baculovirus infection.

Phallotoxins for Labeling F-ActinWe prepare a number of �uorescent and biotinylated derivatives of

phalloidin and phallacidin for selectively labeling F-actin. Phallotoxins are bicyclic peptides isolated from the deadly Amanita phalloides mush-room 6 (www.grzyby.pl/gatunki/Amanita_phalloides.htm). �ey can be used interchangeably in most applications and bind competitively to the same sites on F-actin. Table 11.2 lists the available phallotoxin deriva-tives, along with their spectral properties.

A detailed staining protocol is included with each phallotoxin derivative. One vial of the �uorescent phallotoxin contains su�cient reagent for staining ~300 microscope slide preparations; one vial of biotin-XX phalloidin, which must be used at a higher concentration, contains su�cient reagent for ~50 microscope slide preparations. We also o�er unlabeled phalloidin (P3457) for blocking F-actin staining by labeled phallotoxins and for promoting actin polymerization.

Table 11.2 Spectral characteristics of Molecular Probes® actin-selective probes.

Cat. No. Actin-Selective Probe Ex/Em * Approximate MW

F-Actin–Selective Probes

A22281 Alexa Fluor® 350 phalloidin 346/446 † 1100

C606 Coumarin phallacidin 355/443 1100

N354 NBD phallacidin 465/536 1040


F432 Fluorescein phalloidin 496/516 † 1175

O7466 Oregon Green® 488 phalloidin 496/520 † 1180

B607 BODIPY® FL phallacidin 505/512 1125

O7465 Oregon Green® 514 phalloidin 511/528 † 1280


R415 Rhodamine phalloidin 540/565 † 1250



B3475 BODIPY® 558/568 phalloidin 558/569 1115



T7471 Texas Red®-X phalloidin 591/608 † 1490



B12382 BODIPY® 650/665 phalloidin 647/661 1200




B7474 Biotin-XX phalloidin NA 1300

P3457 Phalloidin NA 790

G-Actin–Selective Probes

D12371 Alexa Fluor® 488 DNase I 495/519 >31,000

D12372 Alexa Fluor® 594 DNase I 590/617 >31,000

* Excitation (Ex) and emission (Em) maxima, in nm. Spectra of phallotoxins are either in aqueous bu�er, pH 7–9 (denoted †) or in methanol. Spectra of DNase I conjugates are in aqueous bu�er, pH 7–8. NA = Not applicable.



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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTATAT NT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to 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.


NOTE 11.1

BacMam Gene Delivery and Expression Technology

Figure 1 Schematic representation of BacMam transgene delivery and expression as ex-emplified by Premo™ Halide Sensor (P10229).

Promoter YFP (Venus)

Baculovirus

Premo™ Halide Sensor gene

Endocytotic entry

DNA movesto nucleus Venus gene

transcribed

DNA

mRNA

mRNAtranslated

YFP (Venus)

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.

Baculovirus-Mediated Transduction of Mammalian CellsBacMam technology uses a modified insect cell baculovirus as a

vehicle to efficiently deliver and express genes in mammalian cells with minimum effort and toxicity.ffort and toxicity.ff 1–4 We have combined the BacMam gene de-livery and expression system with genetically encoded Premo™ sensors as well as with genetically encoded CellLight® targeted fluorescent proteins to yield robust and easy-to-use cell-based assays (Figure 1).

BacMam particles carrying the biosensor or targeted fluorescent protein cDNA under the control of the CMV promoter are taken up by endocytosis. The viral DNA traffics to the nucleus where only the CMV promoter–driven gene is transcribed; baculovirus promoters are not recognized by the mammalian transcriptional machinery. Following tran-scription, the biosensor or targeted fluorescent protein mRNA is expressed in the cytosol and cells are soon ready to assay. This process begins within 4–6 hours after transduction and in many cell types is completed after an overnight period.

BacMam 2.0 vectors incorporated in our CellLight® reagents extend the applicability of BacMam-mediated transgene delivery and expres-sion. Cells such as primary neurons that were not amenable to BacMam transduction with version 1.0 (used in the corresponding Organelle Lights™ and Cellular Lights™ reagents) can now be transduced quantitatively in a simple, one-step process. The improved performance is due to inclusion of a pseudotyped capsid protein for more efficient cell entry as well as genetic elements (enhanced CMV promoter and Woodchuck Post-Transcriptional Regulatory Element) that boost expression levels.

Inducible, division-arrested or transient expression systems such as the BacMam system are increasingly methods of choice to decrease variability of expression in cell-based assays. Constitutively expressed ion channels and other cell-surface proteins have been shown to contribute to cell toxicity in some systems, and they may also be subject to clonal drift and other inconsistencies that hamper successful experimentation and screening. Moreover, the BacMam gene delivery and expression system provides a method for simultaneously delivering multiple genes per cell, an important feature when expressing multisubunit proteins.1

Advantages of the BacMam Delivery and Expression System

Baculoviruses have been used extensively for protein production in insect cells for over two decades; however, its use with mammalian cells is relatively new. BacMam technology has opened up new avenues for mammalian cell–based assays in drug discovery applications.3,5 In addition to producing ready-to-use viral stocks, BacMam delivery and expression

technology has many advantages when compared with lipids and other viral delivery methods:

• High transduction efficiency across a broad range of cell types, includ-ing primary and stem cells

• Minimal microscopically observable cytopathic effectsffectsff• 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, homo-geneous 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.



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Properties of Phallotoxin Derivatives�e �uorescent and biotinylated phallotoxin derivatives stain F-actin selectively at nano-

molar concentrations and are readily water soluble, thus providing convenient labels for iden-tifying and quantitating actin in tissue sections, cell cultures or cell-free preparations.7–11 F-actin in live neurons can be e�ciently labeled using cationic liposomes containing �uores-cent phallotoxins, such as BODIPY® FL phallacidin 12 (B607). �is procedure permits the label-ing of entire cell cultures with minimum disruption. Because �uorescent 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 dye-labeled phalloidins, can penetrate the membranes of certain hypoxic cells.13 An extensive study on visualizing the actin cytoskeleton with various �uorescent probes in cell preparations, as well as in live cells, has been published.7

Labeled phallotoxins have similar a�nity for both large and small �laments 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 di�erent species, including plants and animals; and 2) their nonspeci�c staining is negligible; thus, the contrast between stained and unstained areas is high.

Phallotoxins shift actin’s monomer/polymer equilibrium toward the polymer, lowering the critical concentration for polymerization as much as 30-fold.15,16 Furthermore, depo-lymerization 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 12–15 Å and molecular weights below 2000 daltons, a wide variety of actin-binding proteins—including 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 glyc-erinated muscle fibers still contract, and labeled actin filaments still move on solid-phase myosin substrates.17–19

Alexa Fluor® PhalloidinsWe have taken advantage of the outstanding �uorescence characteristics of our Alexa Fluor®

dyes (Section 1.3) to create a series of Alexa Fluor® dye–labeled 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 11.1.4 Microtubules of �xed bovine pulmonary artery endothelial cells localized with mouse monoclo-nal anti–α-tubulin antibody (A11126), which was subse-quently visualized with Alexa Fluor® 350 goat anti–mouse IgG antibody (A11045). Next, the F-actin was labeled with Alexa Fluor® 594 phalloidin (A12381). Finally, the cells were incubated with Alexa Fluor® 488 wheat germ agglutinin (W11261) to stain components of endosomal pathways. The superimposed and pseudocolored images were acquired sequentially using bandpass �lter sets appropriate for DAPI, the Texas Red® dye and �uorescein, respectively.

Figure 11.1.5 Actin �laments of the turbellarian �atworm Archimonotresis sp. stained with Alexa Fluor® 488 phalloidin (A12379) to reveal a meshwork of longitudinal, circular and diagonal muscles. The large, bright ring with muscle �bers radiating outward is the muscular pharynx, and the small, bright ring at the posterior is part of the reproductive sys-tem. This epi�uorescence image was contributed by Matthew D. Hooge and Seth Tyler, University of Maine, Orono.

Figure 11.1.6 Subcellular structures in �xed and permea-bilized bovine pulmonary artery endothelial cells visualized with several �uorescent dyes. Filamentous actin (F-actin) was identi�ed with Alexa Fluor® 633 phalloidin (A22284), which is pseudocolored magenta. Intracellular membranes were stained with green-�uorescent DiOC6(3) (D273). Finally, nu-clei were counterstained with blue-�uorescent DAPI (D1306, D3571, D21490). The image was acquired using �lters appro-priate for �uorescein and DAPI and a special �lter (courtesy of Omega® Optical) for the Alexa Fluor® 633 dye, consisting of a narrow band exciter (630DF10), dichroic (640DRLP) and emit-ter (660DF10).

Figure 11.1.7 FluoCells® prepared slide #4 (F24631) con-tains a section of mouse intestine stained with a combina-tion of �uorescent stains. Alexa Fluor® 350 wheat germ ag-glutinin (W11263) is a blue-�uorescent lectin that was used to stain the mucus of goblet cells. The �lamentous actin prevalent in the brush border was stained with red-orange–�uorescent 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 �lter sets appropriate for �uorescein, DAPI and tetramethylrhodamine.



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across the full spectral range. �e Alexa Fluor® phalloidin conjugates (Figure 11.1.8) provide researchers with �uorescent probes that are superior in brightness and photostability to other spectrally similar conjugates tested (Figure 11.1.9). For improved �uorescence detection of F-actin in �xed and permeabilized cells, we encourage researchers to try these �uorescent phalloidins in their actin-labeling protocols. A series of videos showing Alexa Fluor® 488 phalloidin–stained actin 20 is available at the Journal of Cell Biology web site (www.jcb.org/cgi/content/full/150/2/361/DC1).

Oregon Green® PhalloidinsGreen-�uorescent actin stains are popular reagents for labeling

F-actin in �xed and permeabilized cells. Unfortunately, the green-�uorescent �uorescein phalloidin and NBD phallacidin photobleach

Figure 11.1.9 Comparison of the photobleaching rates of the Alexa Fluor® 488 and Alexa Fluor® 546 dyes and the well-known �uorescein and Cy®3 �uorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with (top series) Alexa Fluor® 488 phalloidin (A12379) and mouse monoclonal anti–α-tubulin antibody (A11126) in combination with Alexa Fluor® 546 goat anti–mouse IgG antibody (A11003) or (bottom series) �uorescein phalloidin (F432) and the anti–α-tubulin antibody in combination with a commercially available Cy®3 goat anti–mouse IgG antibody. The pseudocolored im-ages were taken at 30-second intervals (0, 30, 90 and 210 seconds of exposure). The images were acquired with bandpass �lter sets appropriate for �uorescein and rhodamine.

Figure 11.1.10 Simultaneous visualization of F- and G-actin in a bovine pulmonary artery en-dothelial cell (BPAEC) using F-actin–speci�c Oregon Green® 488 phalloidin (O7466) and G-actin–speci�c Texas Red® deoxyribonuclease I. The G-actin appears as di�use red �uorescence that is more intense in the nuclear region where the cell thickness is greater and stress �bers are less dense. The image was obtained by taking multiple exposures through bandpass optical �lter sets appropriate for �uorescein and the Texas Red® dye.

Figure 11.1.11 Photostability comparison for Oregon Green® 514 phalloidin (O7465) and �uorescein phalloidin (F432). CRE BAG 2 �broblasts were �xed with formaldehyde, permeabilized with acetone and then stained with the �uo-rescent phallotoxins. Samples were continuously illuminat-ed and images were acquired every 5 seconds using a Star 1 CCD camera (Photometrics); the average �uorescence in-tensity in the �eld of view was calculated with Image-1 soft-ware (Universal Imaging Corp.) and expressed as a fraction of the initial intensity. Three data sets, representing di�erent �elds of view, were averaged for each labeled phalloidin to obtain the plotted time courses.

Fluo

resc

ence

(% o

f ini

tial)

Time (seconds)0 20 40 60

100

80

60

40

20

080 100

Oregon Green® 514

Fluorescein

Figure 11.1.8 Alexa Fluor® 488 phalloidin (A12379).

CH3CH C NHO

NH

CO

CH

NH

C NHO

CH

N C CHO

NH

SH2C

H2C

CO

CH NH C

CHCH3

C

NH

O

OCHCH3

OHHO

CH2

CH3CCH2NHOH

H2N O NH2

CO

O

SO3SO3

6

5

CO

rapidly, making their photography di�cult. We have used two of our Oregon Green® dyes (Section 1.5) to prepare Oregon Green® 488 phalloi-din (O7466, Figure 11.1.10) and the slightly longer-wavelength Oregon Green® 514 phalloidin (O7465). �e excitation and emission spectra of the Oregon Green® 488 dye are virtually superimposable on those of �u-orescein, and both the Oregon Green® 488 and Oregon Green® 514 dyes may be viewed with standard �uorescein optical �lter sets. As shown in Figure 11.1.11, Oregon Green® 514 phalloidin is more photostable than �uorescein phalloidin, making it easier to visualize and photograph.




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BODIPY® PhallotoxinsBODIPY® phallotoxin conjugates (B607, B3475, B12382; Figure 11.1.12, Figure 11.1.13) have

some important advantages over the conventional NBD, �uorescein and rhodamine phallotox-ins. BODIPY® dyes are more photostable than these traditional �uorophores 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 �uorescein phalloidin,22 has been used for quantitating F-actin and determining its distribu-tion in cells.23,24

�e BODIPY® FL and BODIPY® 558/568 phallotoxins (B607, B3475) exhibit excitation and emission spectra similar to those of �uorescein and rhodamine B, respectively, and can be used with standard optical �lter sets. BODIPY® 650/665 phalloidin (B12382) is the longest-wavelength 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 and Other Red-Fluorescent PhalloidinsRhodamine phalloidin (R415, Figure 11.1.14) has been the standard for red-�uorescent phal-

lotoxins.25–27 Rhodamine phalloidin is excited e�ciently by the mercury-arc lamp in most �uo-rescence 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-�uorescent Alexa Fluor® 488, Oregon Green® and �uorescein dyes.

Other Labeled Phallotoxins�e original yellow-green–�uorescent NBD phallacidin (N354) and green-�uorescent

�uorescein phalloidin (F432) remain in use despite their relatively poor photostability (Figure 11.1.11). Photostability of �uorescein phalloidin and some other �uorescent phallotoxins can be considerably improved (Figure 11.1.17) by mounting the stained samples with our ProLong® Antifade Kit or ProLong® Gold antifade reagent (P7481, P36930, P36934; Section 23.1). We rec-ommend the Alexa Fluor® 488, Oregon Green® 488, Oregon Green® 514 and BODIPY® FL phal-lotoxins for photostable, green-�uorescent actin staining. Alexa Fluor® 350 phalloidin (A22281) and coumarin phallacidin (C606, Figure 11.1.2) are the only blue-�uorescent phallotoxin conju-gates currently available for staining actin.29

Biotin-XX phalloidin (B7474) permits detection of F-actin by electron microscopy and light microscopy techniques.30 �is biotin conjugate can be visualized with �uorophore- or enzyme-labeled avidin and streptavidin (Section 7.6) or with tyramide signal ampli�cation (TSA™) tech-nology (Section 6.2). Biotin-XX phalloidin, in conjunction with streptavidin or CaptAvidin™ agarose (S951, C21386; Section 7.6), can be used to precipitate F-actin from the cytosolic anti-phosphotyrosine–reactive fraction in macrophages stimulated with colony-stimulating factor-1.31

DNase I Conjugates for Staining G-ActinBovine pancreatic deoxyribonuclease (DNase I, ~31,000 daltons) binds much more strongly

to monomeric G-actin than to �lamentous F-actin, with binding constants of 5 × 108 M–1 and 1.2 × 104 M–1, respectively.32–35 Because of this strong, selective binding to G-actin, �uorescent 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 �uorescein DNase I, BODIPY® 581/591 phalloidin and a monoclonal anti-actin antibody detected with a Cascade Blue® dye–labeled secondary antibody 36 (C962, Section 7.2). We found that the monoclonal an-tibody, which binds to both G-actin and F-actin, colocalized with the DNase I and phalloi-din 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-�uorescent Alexa Fluor® 488 (D12371) or red-�uorescent Alexa Fluor® 594 (D12372) dyes, depending on their multicolor application and their detection instrumentation (Table 11.2).

Figure 11.1.12 Permeabilized bovine pulmonary artery en-dothelial cells stained with SYTOX® Green nucleic acid stain (S7020) to label the nuclei and with BODIPY® TR-X phallaci-din (B7464) to label the F-actin. The image was acquired by taking sequential exposures through bandpass optical �lter sets appropriate for �uorescein and the Texas Red® dye.

Figure 11.1.13 Actin labeled with BODIPY® FL phallacidin (B607) and vinculin, a cytoskeletal focal adhesion protein, tagged with a monoclonal anti-vinculin antibody that was subsequently probed with Texas Red® goat anti–mouse IgG an-tibody (T862). The large triangular cell is a �broblast containing green actin stress �bers terminating in red focal adhesions. The neighboring polygonal cell, a rat neonatal cardiomyocyte, con-tains green striated actin in the myo�brils terminating in the fo-cal adhesions. The close apposition of the two stains results in a yellowish-orange color. Image contributed by Mark B. Snuggs and W. Barry VanWinkle, University of Texas, Houston.

Figure 11.1.14 Actin �laments of chick heart �broblasts stained with rhodamine phalloidin (R415). The subcompart-ments in the cytoskeleton are readily apparent and labeled as follows: sf, stress �ber; lam, lamellipodium; �l/ms, �lipo-dium/microspike; am, actin meshwork; arc, dorsal arc. Figure reprinted from "Visualizing the Actin Cytoskeleton." J. Small et al. Microscopy Research and Technique (1999) 47:3. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., and J. Victor Small.



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Alexa Fluor® 488 and Alexa Fluor® 594 DNase I conjugates have been used in combination with �uorescently labeled phallotoxins to simultaneously visualize G-actin pools and �lamen-tous F-actin 37,38 and to study the disruption of micro�lament 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 �broblast 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). �e in�uence of cyto-chalasins on actin structure in monocytes has been quantitated by �ow cytometry using Texas Red® DNase I and BODIPY® FL phallacidin (B607) to stain the G-actin and F-actin pools, re-spectively.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

Probes for Actin Quantitation, Actin Polymerization and Actin-Binding ProteinsAssays for Quantitating F-Actin and G-Actin Polymerization

Quantitative assays for F-actin have employed �uorescein phalloidin,43,44 rhodamine phal-loidin,45 BODIPY® FL phallacidin 24 and NBD phallacidin.46 An F-actin assay based on �uores-cein phalloidin was used to demonstrate the loss of F-actin from cells during apoptosis.47 �e ad-dition 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. �e change in F-actin content in proliferating adherent cells has been quanti-tated using the ratio of rhodamine phalloidin �uorescence to ethidium bromide �uorescence.48 �e spectral separation of the signals in this assay may be improved by using a green-�uorescent stain for F-actin and a high-a�nity red-�uorescent nucleic acid stain, such as the combination of Alexa Fluor® 488 phalloidin (A12379) and ethidium homodimer-1 (E1169, Section 8.1).

�e �uorescence of actin monomers labeled with pyrene iodoacetamide (P29) has been dem-onstrated to change upon polymerization, making this probe an excellent tool for following the kinetics of actin polymerization and the e�ects of actin-binding proteins on polymerization.49–51

Figure 11.1.15 A section of mouse intestine stained with a combination of �uorescent stains. Fibronectin, an extracel-lular matrix adhesion molecule, was labeled using a chicken primary antibody against �bronectin and visualized using green-�uorescent Alexa Fluor® 488 goat anti–chicken IgG antibody (A11039). The �lamentous actin (F-actin) preva-lent in the brush border was stained with red-�uorescent Alexa Fluor® 568 phalloidin (A12380). Finally, the nuclei were stained with DAPI (D1306, D3571, D21490).

Figure 11.1.16 Confocal micrograph of the cytoskel-eton of a mixed population of granule neurons and glial cells. The F-actin was stained with red-�uorescent Texas Red®-X phalloidin (T7471). The microtubules were de-tected with a mouse monoclonal anti–ß-tubulin primary antibody and subsequently visualized with the green-�u-orescent Alexa Fluor® 488 goat anti–mouse IgG antibody (A11001). The image was contributed by Jonathan Zmuda, Immunomatrix, Inc.

Figure 11.1.17 Bovine pulmonary artery endothelial cells were labeled with �uorescein phalloidin (F432), which labels �la-mentous actin, and placed under constant illumination on the microscope with a FITC �lter set using a 60× objective. Images were acquired at one-second intervals for 30 seconds. Under these illumination conditions, �uorescein 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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Jasplakinolide: A Cell-Permeant F-Actin ProbeWe o�er jasplakinolide (J7473, Figure 11.1.18), a macrocyclic peptide isolated from the

marine sponge Jaspis johnstoni.52–54 Jasplakinolide is a potent inducer of actin polymerization in vitro by stimulating actin �lament 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. �is peptide, which also exhibits fun-gicidal, insecticidal and antiproliferative activity,53,58–60 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

Latrunculin A and Latrunculin B: Cell-Permeant Actin AntagonistsLatrunculins are powerful disruptors of micro�lament organization. Isolated from a Red Sea

sponge, these G-actin binding compounds inhibit fertilization and early embryological devel-opment,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 con-centrations (Howard Petty, Wayne State University, personal communication) and is frequently used to establish the e�ects of F-actin disassembly on particular physiological functions such as ion transport 67 and protein localization.68 �e activity of latrunculin B (L22290) mimics that of latrunculin A in most applications.63,65,69–71

Assays for Actin-Binding ProteinsEnhancement of the �uorescence of certain phallotoxins upon binding to F-actin can be a

useful tool for following the kinetics and extent of binding of speci�c actin-binding proteins. We have used the change in �uorescence of rhodamine phalloidin (R415) to determine the dissocia-tion constant of various phallotoxins.72 �e enhancement of rhodamine phalloidin’s �uorescence upon actin binding has also been used to measure the kinetics and extent of gelsolin severing of actin �laments.73 �e a�nity and rate constants for rhodamine phalloidin binding to actin are not a�ected by saturation of actin with either myosin subfragment-1 or tropomyosin, indicating that these two actin-binding proteins do not bind to the same sites as the phalloidin.12

Figure 11.1.18 Jasplakinolide (J7473).

Figure 11.1.19 Latrunculin A (L12370).

REFERENCES1. 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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DATA TABLE 11.1 PROBES FOR ACTINCat. No. MW Storage Soluble Abs EC Em Solvent NotesA12379 ~1320 F,L MeOH, H2O 494 78,000 517 pH 7 1, 2, 3A12380 ~1590 F,L MeOH, H2O 578 88,000 600 pH 7 1, 2, 3A12381 ~1620 F,L MeOH, H2O 593 92,000 617 pH 7 1, 2, 3A22281 ~1100 F,L MeOH, H2O 346 17,000 446 pH 7 1, 2, 3A22282 ~1350 F,L MeOH, H2O 528 81,000 555 pH 7 1, 2, 3A22283 ~1800 F,L MeOH, H2O 554 112,000 570 pH 7 1, 2, 3A22284 ~1900 F,L MeOH, H2O 621 159,000 639 MeOH 1, 2, 3, 4A22285 ~1650 F,L MeOH, H2O 668 132,000 697 MeOH 1, 2, 3, 4A22286 ~1850 F,L MeOH, H2O 684 183,000 707 MeOH 1, 2, 3, 4A22287 ~1950 F,L MeOH, H2O 650 275,000 672 MeOH 1, 2, 3, 4A34054 ~1800 F,L MeOH, H2O 622 145,000 640 MeOH 1, 2, 3, 4A34055 ~1900 F,L MeOH, H2O 555 155,000 572 MeOH 1, 2, 3B607 ~1160 F,L MeOH, H2O 505 83,000 512 MeOH 1, 2, 3B3475 ~1115 F,L MeOH, H2O 558 85,000 569 MeOH 1, 2, 3B7474 ~1300 F MeOH, H2O <300 none 1, 2B12382 ~1200 F,L MeOH 647 102,000 661 MeOH 1, 3, 5C606 ~1100 F,L MeOH, H2O 355 16,000 443 MeOH 1, 2, 3F432 ~1175 F,L MeOH, H2O 496 84,000 516 pH 8 1, 2, 3J7473 709.68 F,D MeOH 278 8000 none MeOHL12370 421.55 F,D DMSO <300 noneL22290 395.51 F,D DMSO <300 noneN354 ~1040 F,L MeOH, H2O 465 24,000 536 MeOH 1, 2, 3O7465 ~1280 F,L MeOH, H2O 511 85,000 528 pH 9 1, 2, 3O7466 ~1180 F,L MeOH, H2O 496 86,000 520 pH 9 1, 2, 3P29 385.20 F,D,L DMF, DMSO 339 26,000 384 MeOH 6, 7P3457 ~790 F MeOH, H2O <300 see Notes 2, 8R415 ~1250 F,L MeOH, H2O 542 85,000 565 MeOH 1, 2, 3, 9T7471 ~1490 F,L MeOH, H2O 583 95,000 603 MeOH 1, 2, 3, 9For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. α-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

dye–peptide 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 �uorescent 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 LIST 11.1 PROBES FOR ACTINCat. No. Product QuantityA12375 actin from rabbit muscle 1 mgA12373 actin from rabbit muscle, Alexa Fluor® 488 conjugate *in solution* 200 µgA12374 actin from rabbit muscle, Alexa Fluor® 568 conjugate *in solution* 200 µgA34050 actin from rabbit muscle, Alexa Fluor® 594 conjugate *in solution* 200 µgA34051 actin from rabbit muscle, Alexa Fluor® 647 conjugate *in solution* 200 µgA22281 Alexa Fluor® 350 phalloidin 300 UA12379 Alexa Fluor® 488 phalloidin 300 UA22282 Alexa Fluor® 532 phalloidin 300 UA22283 Alexa Fluor® 546 phalloidin 300 UA34055 Alexa Fluor® 555 phalloidin 300 UA12380 Alexa Fluor® 568 phalloidin 300 UA12381 Alexa Fluor® 594 phalloidin 300 UA22284 Alexa Fluor® 633 phalloidin 300 UA34054 Alexa Fluor® 635 phalloidin 300 UA22287 Alexa Fluor® 647 phalloidin 300 UA22285 Alexa Fluor® 660 phalloidin 300 UA22286 Alexa Fluor® 680 phalloidin 300 UB7474 biotin-XX phalloidin 50 UB3475 BODIPY® 558/568 phalloidin 300 UB12382 BODIPY® 650/665 phalloidin 300 UB607 BODIPY® FL phallacidin 300 UC10582 CellLight® Actin-GFP *BacMam 2.0* 1 mLC10583 CellLight® Actin-RFP *BacMam 2.0* 1 mLC10615 CellLight® Null (control) *BacMam 2.0* 1 mLC606 coumarin phallacidin 300 UD12371 deoxyribonuclease I, Alexa Fluor® 488 conjugate 5 mgD12372 deoxyribonuclease I, Alexa Fluor® 594 conjugate 5 mgF432 �uorescein phalloidin 300 UJ7473 jasplakinolide 100 µgL12370 latrunculin A 100 µgL22290 latrunculin B 100 µgN354 N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD phallacidin) 300 UO7466 Oregon Green® 488 phalloidin 300 UO7465 Oregon Green® 514 phalloidin 300 UP3457 phalloidin 1 mgP29 N-(1-pyrene)iodoacetamide 100 mgR415 rhodamine phalloidin 300 UT7471 Texas Red®-X phalloidin 300 U



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Section 11.2 Probes for Tubulin and Other Cytoskeletal Proteins

11.2 Probes for Tubulin and Other Cytoskeletal Proteins

Figure 11.2.2 Paclitaxel, Oregon Green® 488 conjugate (Oregon Green® 488 Taxol®, Flutax-2; P22310).

Figure 11.2.1 TubulinTracker™ Green (Oregon Green® 488 Taxol®, bis-acetate; T34075).

O

C O

OCCH3CH3CO

O FF

O

CH3

CH3

OCH3

O

C O

CH3

HOO

CO

H3C

O

OO

C O

CH3

C

O

CH2CH2NHOC

O

NHCHCH

OH

C

O

C

OO

Paclitaxel ProbesPaclitaxel

We o�er paclitaxel (P3456) for research purposes only at a purity of >98% by HPLC. Paclitaxel, formerly referred to as taxol in some scienti�c literature, is the approved generic name for the anticancer pharmaceutical Taxol® (Bristol-Myers Squibb Co.). �e diterpenoid paclitaxel is a potent anti-neoplastic agent 1,2 originally isolated from the bark and needles of the western yew tree, Taxus brevifolia.3,4 �e anti-mitotic and cytotoxic action of paclitaxel is related to its ability to promote tubulin assembly into stable aggregated structures that cannot be depolymerized by dilution, calcium ions, cold or a num-ber of microtubule-disrupting drugs; 5–7 paclitaxel also decreases the critical concentration of tubulin required for microtubule assem-bly. Cultured cells treated with paclitaxel are blocked in the G2 (the "gap" between DNA synthesis and mitosis) and M (mitosis) phases of the cell cycle.8

TubulinTracker™ Green ReagentTubulinTracker™ Green reagent (T34075) provides green-�uores-

cent staining of polymerized tubulin in live cells.9–11 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, non�uorescent 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 nonspeci�c esterases, resulting in a green-�uorescent, charged paclitaxel.

TubulinTracker™ Green reagent is provided as a set of two compo-nents: 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.

Fluorescent Paclitaxel ConjugatesIn addition to unlabeled paclitaxel and TubulinTracker™ Green re-

agent, we provide three �uorescent derivatives of paclitaxel: Oregon Green® 488 paclitaxel (Flutax-2, P22310), BODIPY® FL paclitaxel (P7500) and BODIPY® 564/570 paclitaxel (P7501). �ese �uorescent paclitaxel derivatives are promising tools for imaging microtubule formation and motility. �eir �uorescent attributes should also make these conjugates useful reagents for screening compounds that a�ect microtubule assembly.

Oregon Green® 488 paclitaxel 12–16 is an important probe for label-ing tubulin �laments in live cells. �e �uorescent label on this probe is attached by derivatizing the 7β-hydroxy group of native paclitaxel (Figure 11.2.2), a strategy that permits selective binding of the probe to microtubules with high a�nity at 37°C 16 (Kd ~10–7 M). Oregon Green® 488 paclitaxel has been utilized in a high-throughput �uorescence po-larization–based assay to screen for paclitaxel biomimetics.14 We have successfully used Oregon Green® 488 paclitaxel to label microtubules




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Figure 11.2.3 Microtubules were assembled, stabilized and visualized with the aid of green-�uorescent Oregon Green® 488 paclitaxel (P22310). Viable HeLa cells were incubated with the conjugate for 1 hour, followed by several washes with phosphate-bu�ered saline containing 2% bovine serum albumin. The image was acquired using a confocal laser-scanning micro-scope and a �lter set appropriate for �uorescein.

Figure 11.2.4 Paclitaxel, BODIPY® FL conjugate (BODIPY® FL Taxol®, P7500).

Figure 11.2.5 Human mesenchymal stem cell labeled with CellLight® MAP4-GFP (C10598) and CellLight® Histone 2B-RFP (C10595) reagents.

Figure 11.2.6 Microtubules of bovine pulmonary artery endothelial cells tagged with mouse monoclonal anti–α-tubulin an-tibody (A11126) and subsequently probed with: Alexa Fluor® 488 goat anti–mouse IgG antibody (A11001, left), Alexa Fluor® 546 goat anti–mouse IgG antibody (A11003, middle) or Alexa Fluor® 594 goat anti–mouse IgG antibody (A11005, right). These images were acquired using a �uorescein bandpass optical �lter set, a rhodamine bandpass optical �lter set and a Texas Red® bandpass optical �lter set, respectively.

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 re-active �uorophore, a published method describes the use of these BODIPY® paclitaxel derivatives to generate �uorescent 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 ProbesGFP- and RFP-Labeled Tubulin and MAP4

GFP–tubulin fusions are well-established probes for imaging cy-tokinesis and other dynamic rearrangements of microtubules in live cells.20 CellLight® Tubulin-GFP and CellLight® Tubulin-RFP expres-sion vectors (C10613, C10614; Table 11.1) generate auto�uorescent pro-teins fused to the N-terminus of human β-tubulin and incorporate all the generic advantages of BacMam 2.0 delivery technology (BacMam Gene Delivery and Expression Technology—Note 11.1).

In context-speci�c instances where GFP–tubulin fusion protein incorporation into microtubules is ine�cient, 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.



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Anti–α-Tubulin Monoclonal AntibodyWhen used in conjunction with an anti–mouse IgG secondary im-

munoreagent (Section 7.2, Table 7.1), our anti–α-tubulin monoclonal antibody (A11126) enables researchers to visualize microtubules in �xed cells (Figure 11.2.6, Figure 11.2.7, Figure 11.2.8, Figure 11.2.9) and in �xed or frozen tissue sections from various species. �is mouse monoclonal antibody, which recognizes amino acid residues 69–97 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 α-tubulin.

�e anti–α-tubulin monoclonal antibody is available either unla-beled (A11126) or as a biotin-XX conjugate (A21371). For detecting the

Figure 11.2.7 Microtubules of �xed bovine pulmonary artery endothelial cells were labeled with our mouse monoclonal anti–α-tubulin antibody (A11126), detected with the biotin-XX–con-jugated F(ab’)2 fragment of goat anti–mouse IgG antibody (B11027) and visualized with Alexa Fluor® 488 streptavidin (S11223). The actin �laments were then labeled with orange-�uorescent Alexa Fluor® 568 phalloidin (A12380), and the cell was counterstained with blue-�uorescent Hoechst 33342 (H1399, H3570, H21492) to image the DNA, and red-�uorescent propidium iodide (P1304MP, P3566, P21493) to image the nucleolar RNA. The multiple-exposure image was acquired using bandpass �lters appropriate for the Texas Red® dye, �uorescein 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–α-tubulin antibody (A11126) in combination with Alexa Fluor® 594 dye–conjugated F(ab’)2 fragment of goat anti–mouse IgG antibody (A11020) to stain microtubules. The multiple-exposure image was acquired using bandpass �lter sets appropriate for Texas Red® dye and �uorescein.

Figure 11.2.9 A zebra�sh cryosection incubated with the biotin-XX conjugate of mouse monoclonal anti–α-tubulin antibody (A21371). The signal was ampli�ed with TSA™ Kit #22, which includes HRP–streptavidin and Alexa Fluor® 488 tyramide (T20932). The sample was then incubated with the mouse monoclonal FRet 6 antibody and was visualized with Alexa Fluor® 647 goat anti–mouse IgG (A21235), which is pseudocolored magenta. Finally, the nuclei were counter-stained with SYTOX® Orange nucleic acid stain (S11368).

Figure 11.2.10 Fixed and permeabilized bovine pulmo-nary artery endothelial cells stained with Alexa Fluor® 350 phalloidin (A22281), an anti–α-tubulin antibody (A11126) and the anti–cdc6 peptide antibody (A21286). The anti–α-tubulin antibody was labeled with the Zenon® Alexa Fluor® 568 Mouse IgG1 Labeling Kit (Z25006) and the anti–cdc6 peptide antibody was labeled with the Zenon® Alexa Fluor® 488 Mouse IgG1 Labeling Kit (Z25002).

Figure 11.2.11 A prometaphase muntjac skin �broblast stained with Alexa Fluor® 350 phalloidin (A22281), an anti–α-tubulin antibody (A11126) and an anti–cdc6 pep-tide antibody (A21286). The anti–α-tubulin antibody was prelabeled with the Zenon® Alexa Fluor® 488 Mouse IgG1 Labeling Kit (Z25002) and the anti–cdc6 peptide antibody was prelabeled with the Zenon® Alexa Fluor® 647 Mouse IgG1 Labeling Kit (Z25008).

biotinylated antibody, we carry a wide variety of �uorophore- and en-zyme-labeled avidin, streptavidin and NeutrAvidin™ biotin-binding pro-tein conjugates and NANOGOLD® and Alexa Fluor® FluoroNanogold™ streptavidin (Section 7.6, Table 7.9).

We have extensively utilized the mouse IgG1 monoclonal anti–α-tubulin antibody during development and evaluation of our Zenon® technology (Section 7.3, Table 7.7), which allows labeling of submi-crogram quantities of primary antibodies in minutes (Figure 11.2.10, Figure 11.2.11). A comprehensive listing of our primary antibod-ies for cytoskeletal proteins can be found at www.invitrogen.com/handbook/antibodies.




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BODIPY® FL VinblastineBODIPY® FL vinblastine (V12390, Figure 11.2.12), a �uorescent

analog of the anticancer drug vinblastine, is a useful probe for label-ing β-tubulin and for investigating drug-transport mechanisms.21,22 Vinblastine inhibits cell proliferation by capping microtubule ends, thereby suppressing mitotic spindle microtubule dynamics.23 Another �uorescent vinblastine derivative, vinblastine 4 -́anthranilate, report-edly binds to the central portion of the primary sequence of β-tubulin and inhibits polymerization.21,24–26

In addition, intracellular accumulation of vinblastine has been as-sociated with a vinblastine-speci�c modulating site on P-glycoprotein, a drug-e�ux pump that is overexpressed in multidrug-resistant (MDR) cells 27 (Section 15.6). �is highly lipophilic P-glycoprotein substrate has also been used to study the role of P-glycoprotein in drug penetra-tion through the blood-brain barrier.28 Fluorescently labeled vinblas-tine analogs, including BODIPY® FL vinblastine, have been employed to measure drug-transport kinetics in MDR cells.29

Other Probes for Tubulin�e nuclear stain DAPI (D1306, D3571, D21490) binds tightly to

puri�ed tubulin in vitro without interfering with microtubule assembly or GTP hydrolysis. DAPI binds to tubulin at sites di�erent from those of paclitaxel, colchicine and vinblastine, and its binding is accompanied by shi�s in the absorption spectra and �uorescence enhancement. �e a�nity of DAPI for polymeric tubulin is 7-fold greater than for dimeric tubulin, making DAPI a sensitive tool for investigating microtubule assembly kinetics.30–33 DAPI has been used to screen for potential anti-microtubule drugs in a high-throughput assay.34

Bis-ANS (B153) is a potent inhibitor of in vitro microtubule as-sembly.35 �is �uorescent probe binds to the hydrophobic cle�s of proteins with an a�nity approximately 10–100 times higher than that of 1,8-ANS (A47, Section 13.5) and exhibits a signi�cant �uorescence enhancement upon binding. �e bis-ANS binding site on tubulin lies near the critical contact region for microtubule assembly, but it is dis-tinct from the binding sites for colchicine, vinblastine, podophyllo-toxin and maytansine.36–38 Bis-ANS was used to investigate structural changes in tubulin monomers and dimers during time- and tempera-ture-dependent decay.39,40

Figure 11.2.12 Vinblastine, BODIPY® FL conjugate (BODIPY® FL vinblastine, V12390). Figure 11.2.13 HeLa cell labeled with CellLight® Talin-GFP (C10611) and CellLight® Actin-RFP (C10583) reagents.

DCVJ (4-(dicyanovinyl)julolidine; D3923), which binds to a speci�c site on the tubulin dimer,41 has been reported to be a useful probe for following polymerization of tubulin in live cells.42 DCVJ staining in live cells is mostly blocked by cytochalasin D.43 Additionally, DCVJ emits strong green �uorescence upon binding to bovine brain calmodulin.44 �e hydrophobic surfaces of tubulin have also been investigated with the environment-sensitive probes nile red 45 (N1142) and prodan 46 (P248).

Probes for Other Cytoskeletal ProteinsGFP- and RFP-Labeled Talin

Talin is a cytoskeletal protein that is concentrated in focal ad-hesions, linking integrins to the actin cytoskeleton either directly or indirectly by interacting with vinculin and α-actinin. CellLight® Talin-GFP and CellLight® Talin-RFP expression vectors (C10611, C10612; Table 11.1; Figure 11.2.13) generate autof luorescent pro-teins 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 integrin-mediated cell adhesion, as well as for general-purpose labeling of cytoskeletal actin in live cells.

Anti–Glial Fibrillary Acidic Protein (GFAP) Antibody�e 50,000-dalton type-III intermediate �lament protein known as

glial �brillary acidic protein (GFAP) is a major structural component of astrocytes and some ependymal cells.47 GFAP associates with the calcium-binding protein annexin II2-p11(2) and S-100.48,49 Association with these proteins together with phosphorylation regulates GFAP po-lymerization. Astrocytes respond to brain injury by proliferation (as-trogliosis); one of the �rst events to occur during astrocyte prolifera-tion is increased GFAP expression. Our anti-GFAP antibody (A21282) and its Alexa Fluor® 488 and Alexa Fluor® 594 conjugates (A21294, A21295; Figure 11.2.14) can be used to aid in the identi�cation of cells of glial lineage. Interestingly, antibodies to GFAP have been detected in



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individuals with dementia.50 In the central nervous system, anti-GFAP antibody stains both astrocytes and ependymal cells. In the periph-eral nervous system, Schwann cells, satellite cells and enteric glial cells are stained; tumors of glial origin contain high amounts of GFAP. No positive staining is observed in skin, connective tissue, adipose tissue, lymphatic tissue, muscle, kidney, ureter, bladder or gastrointestinal tract, including liver and pancreas. Our anti-GFAP antibody does not cross-react with vimentin, which is frequently co-expressed in glioma cells and some astrocytes, nor does it cross-react with Bergmann glia cells, gliomas or other glial cell–derived tumors.

Anti-Desmin AntibodyDesmin, encoded by a gene belonging to the intermediate �lament

protein gene family,51–53 is the main intermediate �lament in mature skeletal, cardiac and smooth muscle cells. Both striated and smooth muscle cells can be labeled by an anti-desmin antibody, although not all muscle tissue contains desmin (e.g., aorta smooth muscle). Identi�cation of desmin is useful in distinguishing habdomyosarcomas and leiomyo-sarcomas from other vimentin-positive sarcomas. We o�er a mouse IgG1 monoclonal anti-desmin antibody (A21283), which can be used with our �uorescent secondary antibodies (Section 7.2, Figure 11.2.15) as a marker for typing so� tissue sarcomas. Anti-desmin immunohis-tochemical staining in cell-block preparations may also be helpful in distinguishing mesothelial cells from carcinoma.54

Anti-Synapsin I AntibodySynapsin I, an actin-binding protein, is localized exclusively to

synaptic vesicles and thus serves as an excellent marker for synapses in brain and other neuronal tissues.55,56 Synapsin I inhibits neurotrans-mitter release, an e�ect that is abolished upon its phosphorylation by Ca2+/calmodulin–dependent protein kinase II. For assaying the local-ization and abundance of synapsin I by western blot analysis, immuno-histochemistry (Figure 11.2.16), enzyme-linked immunosorbent assay (ELISA) or immunoprecipitation, we o�er a polyclonal rabbit anti–syn-apsin I antibody as an a�nity-puri�ed IgG fraction (A6442). Although raised against bovine synapsin I, this antibody also recognizes human, rat and mouse synapsin I; it has little or no activity against synapsin II.

Figure 11.2.15 The intermediate �laments in bovine pulmonary artery endothelial cells, local-ized using our anti-desmin antibody (A21283), which was visualized with the Alexa Fluor® 647 goat anti–mouse 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-�uores-cent DAPI (D1306, D3571, D21490).

Figure 11.2.16 Peripheral neurons in mouse intestinal cryosections were labeled with rabbit anti–synapsin I antibody (A6442) and detected using Alexa Fluor® 488 goat anti–rabbit IgG antibody (A11008). This tissue was counterstained with DAPI (D1306, D3571, D21490).

Figure 11.2.14 Rat brain cryosections were labeled with the red-�uorescent Alexa Fluor® 594 conjugate of anti–glial �brillary acidic protein antibody (A21295). Nuclei were counterstained with TOTO®-3 iodide (T3604, pseudocolored blue).




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PRODUCT LIST 11.2 PROBES FOR TUBULIN AND OTHER CYTOSKELETAL PROTEINSCat. No. Product QuantityA21283 anti-desmin, mouse IgG1, monoclonal 131-15014 *1 mg/mL* 100 µLA21282 anti-GFAP (anti–glial �brillary acidic protein, mouse IgG1, monoclonal 131-17719) *1 mg/mL* 100 µLA21294 anti-GFAP, Alexa Fluor® 488 conjugate (anti–glial �brillary acidic protein, mouse IgG1, monoclonal 131-17719, Alexa Fluor® 488 conjugate) *1 mg/mL* 50 µLA21295 anti-GFAP, Alexa Fluor® 594 conjugate (anti–glial �brillary acidic protein, mouse IgG1, monoclonal 131-17719, Alexa Fluor® 594 conjugate) *1 mg/mL* 50 µLA6442 anti-synapsin I (bovine), rabbit IgG fraction *a�nity puri�ed* 10 µgA11126 anti-α-tubulin (bovine), mouse IgG1, monoclonal 236-10501 50 µgA21371 anti-α-tubulin (bovine), mouse IgG1, monoclonal 236-10501, biotin-XX conjugate 50 µgB153 bis-ANS (4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid, dipotassium salt) 10 mgC10598 CellLight® MAP4-GFP *BacMam 2.0* 1 mLC10599 CellLight® MAP4-RFP *BacMam 2.0* 1 mLC10611 CellLight® Talin-GFP *BacMam 2.0* 1 mLC10612 CellLight® Talin-RFP *BacMam 2.0* 1 mLC10613 CellLight® Tubulin-GFP *BacMam 2.0* 1 mLC10614 CellLight® Tubulin-RFP *BacMam 2.0* 1 mLD3923 DCVJ (4-(dicyanovinyl)julolidine) 25 mgD1306 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) 10 mgD21490 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) *FluoroPure™ grade* 10 mgD3571 4’,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate) 10 mgN1142 nile red 25 mgP3456 paclitaxel (Taxol® equivalent) *for use in research only* 5 mgP7501 paclitaxel, BODIPY® 564/570 conjugate (BODIPY® 564/570 Taxol®) 10 µgP7500 paclitaxel, BODIPY® FL conjugate (BODIPY® FL Taxol®) 10 µgP22310 paclitaxel, Oregon Green® 488 conjugate (Oregon Green® 488 Taxol®; Flutax-2) 100 µgP248 prodan (6-propionyl-2-dimethylaminonaphthalene) 100 mgT34075 TubulinTracker™ Green (Oregon Green® 488 Taxol®, bis-acetate) *for live-cell imaging* 1 setV12390 vinblastine, BODIPY® FL conjugate (BODIPY® FL vinblastine) 100 µg

DATA TABLE 11.2 PROBES FOR TUBULIN AND OTHER CYTOSKELETAL PROTEINSCat. No. MW Storage Soluble Abs EC Em Solvent NotesB153 672.85 L pH >6 395 23,000 500 MeOH 1, 2D1306 350.25 L H2O, DMF 342 28,000 450 pH 7 3D3571 457.49 L H2O, MeOH 342 28,000 450 pH 7 3D3923 249.31 L DMF, DMSO 456 61,000 493 MeOH 4D21490 350.25 L H2O, DMF 342 28,000 450 pH 7 3, 5N1142 318.37 L DMF, DMSO 552 45,000 636 MeOH 6P248 227.31 L DMF, MeCN 363 19,000 497 MeOH 7P3456 853.92 F,D MeOH, DMSO 228 30,000 none MeOHP7500 1023.89 FF,D,L DMSO 504 66,000 511 MeOHP7501 1098.98 FF,D,L DMSO 565 121,000 571 MeOHP22310 1319.28 FF,D,L DMSO, EtOH 494 80,000 522 pH 9V12390 1043.02 F,D,L DMSO, DMF 503 83,000 510 MeOHFor de�nitions 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 0.1–1.0 mM after heating.2. Bis-ANS (B153) bound to tubulin has Abs = 392 nm, Em = 490 nm and a �uorescence quantum yield of 0.23. (Biochemistry (1994) 33:11900)3. DAPI undergoes an approximately 9-fold �uorescence enhancement on binding to polymerized tubulin. Abs = 345 nm, Em = 446 nm. (J Biol Chem (1985) 260:2819)4. The absorption and �uorescence emission maxima of DCVJ (D3923) bound to tubulin are essentially the same as in methanol. (Biochemistry (1989) 28:6678)5. This product is speci�ed to equal or exceed 98% analytical purity by HPLC.6. The �uorescence emission maximum of nile red (N1142) bound to tubulin is 623 nm. (J Biol Chem (1990) 265:14899)7. The �uorescence emission maximum of prodan (P248) bound to tubulin is ~450 nm. (Eur J Biochem (1992) 204:127)

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