Acti-stain 488 phalloidin

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).

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Example Results and Specifications

	Figure 1. Actin Stress Fibers stained with Acti-stain™ 488 in a Swiss 3T3 cell.		Figure 2. Emission and excitation scans for Acti-stain™ phalloidins
	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).		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.