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FAQs


About Actin

In the following section we refer to frequently asked questions concerning rabbit skeletal muscle actin. The properties of non-muscle or smooth muscle actins may vary from these descriptions. Please refer to the DataSheets for other than alpha-skeletal muscle actins or email your questions to our CustomerService (cusserv@hypermol.com).  



Question

How stable is actin in solution?


Answer

To maintain the biological activity of actin it is crucial to store F-actin and G-actin solutions on ice (0°C). Use a commercially available ice bucket or any styro box from the lab. This may appear trivial, but we have experienced more often than we thought, that actin has been stored in the fridge. Both, fridge (> + 4°) and freezer (< -20°C) are not the right place to store actin! 

G-actin solutions are stable for about one week. After this actin is still polymerizable, but an increasing number of nuclei can form, which affect actin kinetics. If G-actin solutions are not used in kinetic experiments, the solution can still be used for polymerization and is usually not degraded, if free from bacteria.

Solutions of F-actin are more stable, for about two weeks and longer, provided that bactrial growth is prevented. We recommend to add 0.2 mM ATP, pH 7.4
(100mM ATP, pH 7.4, Cat. #: 5122-01) each week to the solution on ice.
 

 

Question

How can actin solutions be stabilized?


Answer

Actin from Hypermol is usually supplied as a freeze dried powder. Prior to freeze-drying the actin solution is sterile filtered (0.2 µm). Actin should only be reconstituted with ultrapure water (0.2 µm filtered), to avoid unnecessary introduction of bacteria. Actin solutions contain ATP, which is a favorable substrate for bacteria.  

 

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Question

What conditions are needed to polymerize actin?


Answer
Monomeric actin - also called Globular-actin (G-actin) - readily polymerizes under physiological buffer conditions to form actin filaments (F-actin). Actin is an ATPase, since the polymerization process is associated with the hydrolysis of ATP by each actin monomer. During polymerization F-actin forms a double-helical filament.:


 
In vitro actin can polymerize from both ends, whereas both ends have rather different rates of polymerization. As a results actin filaments possess an intrinsic polarity. The fast polymerizing end is termed the plus end and  the slow growing end is termed the minus end.

The propensity of G-actin to polymerize into actin filaments depends on the affinity of the actin monomers for the filament ends. Below a certain actin monomer concentration (0.12µM), actin alone cannot polymerize. This concentration is termed the “
Critical Concentration”, short Cc. At monomer concentrations above the Cc, actin polymerizes until the concentration of the free actin monomers is between to the Cof the plus end and the minus end.
 
When one is working with actin in vitro, the extent of actin polymerization depends upon the solvent conditions. The Cc is a function of the rate constants for monomer association and dissociation and both processes depend on parameters like ion type, concentration and ionic strength:

Cc decreases as the ionic strength increases;
Cdecreases as pH decreases;
Cdecreases as increases; 


Alterations of the ion type (e.g. monovalent: K+ vs. Na+, divalent: Mg++ vs. Ca++) and concentration lead to a variation in quantity and lengths of actin filaments. Magnesium strongly promotes the nucleation of actin compared to Calcium. Thus polymerization with 2 mM Mg++ alone produces a higher quantity of slightly shorter filaments compared to the polymerization with 50-100mM KCl alone. 
 
 
Note that actins from different sources have different Ccs.



Question

 
At what speed shall G-actin be centrifuged prior to use?


Answer
 
This depends on the purpose. If you wish to make actin filaments to work with, actin from Hypermol can be reconstituted and used without any pre-treatments.
 
Actin supplied in a freeze-dried state, dissolves instantly upon addition of water. Nuclei or short oligomers which form as a result of freeze-drying, alter the polymerization kinetics. This holds true for actin and actin-conjugates. Therefore, whenever G-actin is required as a starting material, it is necessary to ensure the G-actin state using one of the following methods:
 
-       Centrifugation at 100.000xg, 3h or 150.000xg, 2h;
-       Gel permeation chromatography (e.g. Superdex 200);


 
 
Question
 
At what speed can F-actin be pelleted?
 

Answer
 
Actin filaments can be centrifuged at 100.000xg for 3h at ~15°C. The  Cc is higher at lower temperature, which results in an increased quantity of free actin monomers. Thus, the yield of actin filaments is higher at moderate temperature.  


 
  

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