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Phalloidin-AMCA Conjugate

Chemical structure for Phalloidin-AMCA Conjugate
Chemical structure for Phalloidin-AMCA Conjugate
Ordering information
Price ()
Catalog Number23100
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1100
SolventDMSO
Spectral properties
Correction Factor (280 nm)0.153
Extinction coefficient (cm -1 M -1)19000
Excitation (nm)346
Emission (nm)434
Storage, safety and handling
H-phraseH301, H311, H331
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR23, R24, R25
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
1100
Correction Factor (280 nm)
0.153
Extinction coefficient (cm -1 M -1)
19000
Excitation (nm)
346
Emission (nm)
434
This blue fluorescent phalloidin conjugate (equivalent to coumarin-labeled phalloidin) selectively binds to F-actins. Used at nanomolar concentrations, phalloidin derivatives are convenient probes for labeling, identifying and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell cultures or cell-free experiments. Phalloidin binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the rate constant for the dissociation of actin subunits from filament ends, essentially stabilizing actin filaments through the prevention of filament depolymerization. Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin. Phalloidin functions differently at various concentrations in cells. When introduced into the cytoplasm at low concentrations, phalloidin recruits the less polymerized forms of cytoplasmic actin as well as filamin into stable "islands" of aggregated actin polymers, yet it does not interfere with stress fibers, i.e. thick bundles of microfilaments. The property of phalloidin is a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain actin filaments for light microscopy. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in living or fixed cells as well as for visualizing individual actin filaments in vitro. Fluorescent phalloidin derivatives have been used as an important tool in the study of actin networks at high resolution. AAT Bioquest offers a variety of fluorescent phalloidin derivatives with different colors for multicolor imaging applications.

Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare samples in microplate wells
  2. Remove liquid from samples in the plate
  3. Add Phalloidin-AMCA Conjugate solution (100 μL/well)
  4. Stain the cells at room temperature for 20 to 90 minutes
  5. Wash the cells
  6. Examine the specimen under microscope with DAPI filter 
Important      Warm the vial to room temperature and centrifuge briefly before opening.

Storage and Handling Conditions
The solution should be stable for at least 6 months if store at -20 °C. Protect the fluorescent conjugates from light, and avoid freeze/thaw cycles.
Note     Phalloidin is toxic, although the amount of toxin present in a vial could be lethal only to a mosquito (LD50 of phalloidin = 2 mg/kg), it should be handled with care.

PREPARATION OF WORKING SOLUTION

Phalloidin-AMCA Conjugate working solution
Add 1 µL of Phalloidin-AMCA Conjugate solution to 1 mL of PBS with 1% BSA.
Note     The stock solution of phalloidin conjugate should be aliquoted and stored at -20 °C. protected from light.
Note     Different cell types might be stained differently. The concentration of phalloidin conjugate working solution should be prepared accordingly.

SAMPLE EXPERIMENTAL PROTOCOL

Stain the cells
  1. Perform formaldehyde fixation. Incubate cells with 3.0–4.0 % formaldehyde in PBS at room temperature for 10–30 minutes.
    Note     Avoid any methanol containing fixatives since methanol can disrupt actin during the fixation process. The preferred fixative is methanol-free formaldehyde.
  2. Rinse the fixed cells 2–3 times in PBS.
  3. Optional: Add 0.1% Triton X-100 in PBS into fixed cells for 3 to 5 minutes to increase permeability. Rinse the cells 2–3 times in PBS.
  4. Add 100 μL/well (96-well plate) of Phalloidin-AMCA Conjugate working solution into the fixed cells, and stain the cells at room temperature for 20 to 90 minutes.
  5. Rinse cells gently with PBS 2 to 3 times to remove excess phalloidin conjugate before plating, sealing and imaging under microscope with DAPI filter set. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Phalloidin-AMCA Conjugate to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM90.909 µL454.545 µL909.091 µL4.545 mL9.091 mL
5 mM18.182 µL90.909 µL181.818 µL909.091 µL1.818 mL
10 mM9.091 µL45.455 µL90.909 µL454.545 µL909.091 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

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Spectrum


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spectrum

Spectral properties

Correction Factor (280 nm)0.153
Extinction coefficient (cm -1 M -1)19000
Excitation (nm)346
Emission (nm)434

Citations


View all 28 citations: Citation Explorer
Tissue-type plasminogen activator regulates p35-mediated Cdk5 activation in the postsynaptic terminal
Authors: Diaz, Ariel and Jeanneret, Valerie and Merino, Paola and McCann, Patrick and Yepes, Manuel
Journal: Journal of cell science (2019): jcs224196
Enhanced bovine serum albumin absorption on the N-hydroxysuccinimide activated graphene oxide and its corresponding cell affinity
Authors: Xiong, Kun and Fan, Qingbo and Wu, Tingting and Shi, Haishan and Chen, Lin and Yan, Minhao
Journal: Materials Science and Engineering: C (2017)
Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic Targeting and Imaging
Authors: Sun, Lu and Xie, Shuping and Qi, Jing and Liu, Ergang and Liu, Di and Liu, Quan and Chen, Sunhui and He, Huining and Yang, Victor C
Journal: ACS Applied Materials & Interfaces (2017)
The correlation between osteopontin adsorption and cell adhesion to mixed self-assembled monolayers of varying charges and wettability
Authors: Hao, Lijing and Li, Tianjie and Yang, Fan and Zhao, Naru and Cui, Fuzhai and Shi, Xuetao and Du, Chang and Wang, Yingjun
Journal: Biomaterials Science (2017)
Mediation of cellular osteogenic differentiation through daily stimulation time based on polypyrrole planar electrodes
Authors: Liu, Zongguang and Dong, Lingqing and Wang, Liming and Wang, Xiaozhao and Cheng, Kui and Luo, Zhongkuan and Weng, Wenjian
Journal: Scientific reports (2017): 17926
Study on the Regulation of Focal Adesions and Cortical Actin by Matrix Nanotopography in 3D Environment
Authors: Han, Jingjing and Lin, Keng-hui and Chew, Lock Yue
Journal: Journal of Physics: Condensed Matter (2017)
Enhanced osteointegration of tantalum-modified titanium implants with micro/nano-topography
Authors: Shi, Junyu and Zhang, Xiaomeng and Qiao, Shichong and Ni, Jie and Mo, Jiaji and Gu, Yingxin and Lai, Hongchang
Journal: RSC Advances (2017): 46472--46479
Microtopography Attenuates Endothelial Cell Proliferation by Regulating MicroRNAs
Authors: Wang, Dan and Liu, Mengya and Gu, Shuangying and Zhou, Yue and Li, Song
Journal: Journal of Biomaterials and Nanobiotechnology (2017): 189--201
Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery
Authors: Chang, Ting-Ya and Chen, Chen and Lee, Min and Chang, Ya-Chu and Lu, Chi-Huan and Lu, Shao-Tzu and Wang, De-Yao and Wang, Aijun and Guo, Chin-Lin and Cheng, Pei-Lin
Journal: eLife (2017): e31101
Grafting of Ring-Opened Cyclopropylamine thin films on Silicon (100) Hydride via UV Photoionization
Authors: Tung, Joline and Ching, Jing Yuan and Ng, Yoke Mooi and Tew, Lih Shin and Khung, Yit Lung
Journal: ACS Applied Materials & Interfaces (2017)

References


View all 127 references: Citation Explorer
Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes
Authors: Lin G, Qiu X, F and el TM, Albersen M, Wang Z, Lue TF, Lin CS.
Journal: Urology (2011): 970 e1
Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin
Authors: Diensthuber RP, Muller M, Heissler SM, Taft MH, Chizhov I, Manstein DJ.
Journal: FEBS Lett (2011): 767
pH-(low)-insertion-peptide (pHLIP) translocation of membrane impermeable phalloidin toxin inhibits cancer cell proliferation
Authors: An M, Wijesinghe D, Andreev OA, Reshetnyak YK, Engelman DM.
Journal: Proc Natl Acad Sci U S A (2010): 20246
Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging
Authors: Chazotte B., undefined
Journal: Cold Spring Harb Protoc (2010): pdb prot4947
Protective effect of bile acid derivatives in phalloidin-induced rat liver toxicity
Authors: Herraez E, Macias RI, Vazquez-Tato J, Hierro C, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 21
Effect of Phalloidin on Filaments Polymerized from Heart Muscle Adp-Actin Monomers
Authors: Vig A, Dudas R, Kupi T, Orban J, Hild G, Lorinczy D, Nyitrai M.
Journal: J Therm Anal Calorim (2009): 721
In vitro inhibition of OATP-mediated uptake of phalloidin using bile acid derivatives
Authors: Herraez E, Macias RI, Vazquez-Tato J, Vicens M, Monte MJ, Marin JJ.
Journal: Toxicol Appl Pharmacol (2009): 13
Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes
Authors: Luo H, Hallen-Adams HE, Walton JD.
Journal: J Biol Chem (2009): 18070
Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations
Authors: Wollesen T, Loesel R, Wanninger A.
Journal: J Neurosci Methods (2009): 63
Anti-acetylated tubulin antibody staining and phalloidin staining in the starlet sea anemone Nematostella vectensis
Authors: Genikhovich G, Technau U.
Journal: Cold Spring Harb Protoc (2009): pdb prot5283