Product Uses Include
- Fluorescent staining of actin filaments in fixed tissue sections and tissue culture cells preparations. Note: Unlike many actin antibodies, Acti-stain™ 488 phalloidin binds only to F-actin resulting in low background fluorescence. Furthermore, binding of F-actin by Acti-stain is not appreciably different between species.
- Preparation of stabilized fluorescent actin filaments in vitro.
Actin staining is very useful in determining the structure and function of the cytoskeleton in living and fixed cells. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed with paraformaldehyde prior to probing or staining for actin structures.
Material
Phalloidin is a seven amino acid peptide toxin from the mushroom Amanita phalloides, which binds specifically and with high affinity (Kd 20 nM) to the polymerized form of actin (F-actin). Phalloidin lowers the critical concentration of actin polymerization to less than 1 µg/ml, thereby acting as a polymerization enhancer. Phalloidin has been labeled with a proprietary green fluorescent dye which allows it to be used to stain actin filaments in tissue cultured cells and tissue sections (1, see Figure 1) and cell-free preparations. Acti-stain™ 488 phalloidin-labeled actin filaments retain many functional characteristics of unlabeled actin including their ability to interact with myosin. Actin-stain™ 488 phalloidin is supplied as an orange solid.
Note: Phalloidin is toxic and must be handled with care (LD50 human = 2mg/Kg).
Example Results and Specifications
Figure 1. Actin Stress Fibers stained with Acti-stain™ 488 in a Swiss 3T3 cell.
Swiss 3T3 cell stained with anti-vinculin (red), Dapi (blue nucleus) and F-actin is stained with Acti-stain™ 488 (green F-actin, Cat.# PHDG1).
Figure 2. Emission and excitation scans for Acti-stain™ phalloidins
Absorbance and fluorescence scan of Acti-stain™ 488. Labeled phalloidin was diluted into methanol and its absorbance and excitation spectra were scanned between 350-750 and 500-750 nm, respectively. Absorbance peaks at 500 nm and fluorescence at 550 nm.
Acti-stain phalloidins are the most well characterized phalloidins available. Tabe 1 describes their brightness, photostability, background and affinity constants to F-actin. Compare these performance characteristics to other fluorescent phalloidins and you will see the advantages of using Acti-stain™ for your actin staining requirements.
Table 1. Biochemical characteristics of fluorescent phalloidins
| Conjugate | Cat.# | Wavelengths (Ex/Em) | Brightness (AFU*) | Stability to photobleaching(half life in seconds**) | Background (% of total AFU at 100nM**) | Affinity (Kd in nanomolar) |
Fluorescein-phalloidin | na | 485/535 FITC filter set | 432 | 6 | 22 | 72 +/-12 |
Acti-stain™ 488 | PHDG1 | 485/535 FITC filter set | 832 | 57 | 5 | 55 +/-8 |
Acti-stain™ 535 | PHDR1 | 535/585 TRITC filter set | 430 | 27 | 12 | 36 +/-7 |
Acti-stain™ 555 | PHDH1 | 535/585 TRITC filter set | 551 | 46 | 16 | 63 +/-8 |
Acti-stain™ 670 | PHDN1 | 640/680 Cy5 filter set | 332 | 8 | 18 | 50 +/-12 |
* = AFU's measured by quantitative cell imaging. ** = Measured in stained Swiss 3T3 cells in the absence of antifade.
For more information about protocols to fix and stain cells click here.
References
1. Wulf, E. et al. (1979). Proc Natl Acad Sci USA. 76(9):4498-4502.
2. Kron, S.J. et al. (1991). Meth. Enzmol. 196: 399-416.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Hamdan, H. et al. Mapping axon initial segment structure and function by multiplexed proximity biotinylation. Nat. Commun. 11, 1–17 (2020).
Musah, A. S. et al. Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination. J. Neurosci. 40, 2993 LP – 3007 (2020).
Gupta, D. et al. Fucoidan Inhibition of Osteosarcoma Cells is Species and Molecular Weight Dependent. Mar. Drugs 18, 104 (2020).
Shindo, T. et al. Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer. J. Biochem. (2020) doi:10.1093/jb/mvaa125.
Musah, Aminat S et al. “Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 40,15 (2020): 2993-3007.
Lu, Chi-Sheng et al. “KC21 Peptide Inhibits Angiogenesis and Attenuates Hypoxia-Induced Retinopathy.” Journal of cardiovascular translational research vol. 12,4 (2019): 366-377.
Zhao, Yan et al. “TCF7L2 and EGR1 synergistic activation of transcription of LCN2 via an ERK1/2-dependent pathway in esophageal squamous cell carcinoma cells.” Cellular signalling vol. 55 (2019): 8-16.
Kommaddi, Reddy Peera et al. “Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 38,5 (2018): 1085-1099.
Roitbak et al., 2011. The role of microRNAs in neural stem cell supported endothelial morphogenesis. Vascular Cell. v 3, p 25.
Yuan et al., 2012. Subsecond absolute quantitation of amine metabolites using isobaric tags for discovery of pathway activation in mammalian cells. Anal. Chem. v 84, pp 2892–2899.
Question 1: Can I use fluorescently-labeled phalloidin to stain F-actin in living cells?
Answer 1: Unfortunately, no, phalloidin cannot be used to stain F-actin in living cells. Phalloidins are used to stain F-actin in fixed cells. Fluorescent phalloidins only bind to the native quaternary structure of F-actin which provides a low background. The correct fixation condition for phalloidin binding is 3.7% (v/v) paraformaldehyde in PBS for 10 min because it retains the quaternary protein structure which is necessary for high affinity binding of phalloidin. Methanol fixation destroys the native conformation and hence is not suitable for F-actin staining with phalloidin. To monitor actin dynamics in living cells, micro-injection of rhodamine-labeled actin (Cat. # APHR or AR05) is recommended. Please see those datasheets for more information.
Question 2: Which of the fluorescently-labeled phalloidin is the most stable/brightest?
Answer 2: The brightest and most stable of the Acti-stains is Acti-stain 488 (green fluorescence; Cat. # PHDG1). Please see the table below for additional information on all of our Acti-stains.
Conjugate | Cat. # | Wavelengths (Ex/EM) | Brightness (AFU) | Stability to photobleaching* (1/2 life in sec) | Background (% of total AFU at 100 nM) |
Acti-stain™ 488 | 485/535 | 832 | 57 | 5 | |
Acti-stain™ 535 | 535/585 | 430 | 27 | 12 | |
Acti-stain™ 555 | 535/585 | 551 | 46 | 16 | |
Acti-stain™ 670 | 640/680 | 332 | 8 | 18 |
* = measured in the absence of antifade.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.
