Tip 1: Storage and handling of actin
Tip 2: Staining F-actin with phalloidin
Question 1: Under what circumstances does actin polymerize?
Question 2: What are the main types of molecular interactions with F-actin?
Question 3: What concentration is intracellular actin?
For more specific technical tips please view our product pages and datasheets.
Tip #1: Keep the lyophilized actin dry by storing in a desiccator at either 4°C or -70°C. After reconstituting actin as directed on the product's datasheet, experiment-sized aliquots must be snap-frozen to liquid nitrogen before storing at -70°C to preserve actin's activity. To maintain activity, actin should not be frozen at a concentration below 10 mg/ml. When thawing the aliquots, thaw tubes in a room temperature water bath and immediately place on ice until used. Do not re-freeze actin once thawed. Throw away unused thawed actin aliquots.
Tip #2: Polymerized actin (aka filamentous or F-actin) is not stable to freezing, but can be stored at 4°C for 2-4 weeks.
Tip #3: Actin monomers are unstable in the absence of ATP, a divalent cation and dithiothreitol (DTT).
Staining F-actin with phalloidin
When preparing cells for phalloidin staining, remember to fix cells in 4% paraformaldehyde rather than methanol. Methanol disrupts the structure of F-actin and the phalloidin binding site.
Under what circumstances does actin polymerize?
Globular-actin (G-actin) readily polymerizes under physiological conditions to form Filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament as shown in Figure 1.
Figure 1. Double-helical structure of actin filaments.
Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end (see Figure 1) or barbed-end while the slow growing end is called the minus-end or pointed-end.
The propensity of actin to polymerize is dependent upon the affinity of actin monomers for filament ends. Thus, there is an actin monomer concentration below which actin will not polymerize. This value has been termed the Critical Concentration (CC). At monomer concentrations above the CC, the actin will polymerize until the free monomer concentration is equal to the CC. When one is working with actin in vitro the extent of actin polymerization depends upon the conditions used. For example, at 4°C muscle actin has a CC of 0.03 mg/ml in the presence of 2 mM Mg2+ and 50 mM KCl, but when these ions are absent, the CC is greater than 3.0 mg/ml. Thus, by altering the ionic type and strength one can alter the amount of polymer formed. Non-muscle actin has its own CC values. For example, at 4°C in the presence of 2 mM Mg2+ and 50 mM KCl the CC is approximately 0.15 mg/ml. If Mg²+ and KCl are replaced with Ca²+, the CC will increase to nearly 10 mg/ml. Finally, the CC of non-muscle actin can be reduced to 0.03 mg/ml by increasing the temperature to 30°C.
What procedure do you recommend for in vitro actin polymerization?
We recommend the following procedure for polymerizing actin:
For more critical work, cold spin at 150,000 x g for 2 h after step 1 to pellet small oligomers of actin and use the top 80% of supernatant for polymerization studies.
What are the main types of molecular interactions with F-actin?
There are different kinds of bonds underlying interactions between molecules. One type of bond is noncovalent bonding and this is further broken down into subtypes. The main subtype of noncovalent binding by actin filaments is ionic-based which means that salt concentraitons higher than 75mM tend to dissociate the interactions. Molecular interactions with F-actin are not usually hydrophobic.
What concentration is intracellular actin?
The concentration of actin in non-muscle cells is typically 2-3 mg/ml (46-70 µM). The concentration in muscle cells is typically 10-30 mg/ml (230-960 µM).