Acti-stain™ 488 phalloidin

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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-C) 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)

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.

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, 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.