Actin Polymerization Biochem Kit

Question 1: Can I use this polymerization kit to measure polymerization of non-muscle actin?

Answer 1:  Pyrene-labeled non-muscle actin has been shown to be unstable under normal storage conditions.  To examine the polymerization of unlabeled non-muscle actin, please click here for a polymerization protocol that uses an excess of unlabeled non-muscle, cardiac or gizzard actin (Cat# APHL95, APHL99, AS99) + a small amount of pyrene-labeled skeletal muscle actin (Cat. # AP05).  The pyrene muscle actin will not polymerize efficiently on its own at the concentration used in this assay, so the reaction is dependent on unlabeled actin polymerization for F-actin formation.  In this way, the pyrene-labeled muscle actin is taken up and polymerized to serve as a reporter for polymerization of the unlabeled actin that is present at a much greater concentration. 

Question 2:  Can cell lysates be used with this kit as the source of a test protein?

Answer 2:   Yes, cell lysates can be used as the source of the test protein for examining actin polymerization enhancers and inhibitors.  However, Cytoskeleton does not recommend this as the purity and concentration of the protein will often be too low to interact with actin.  Also, the lysates will contain additional accessory proteins and multiple phosphatases and proteases that can interfere or alter the interactions between actin and test protein.  If lysates are to be used, we recommend the following:

Although this kit is designed for use with pure proteins or compounds, some researchers have added extracts with good results. Generally researchers use over-expressed proteins and a wild-type control extract similarly over-expressed.  It is necessary to make a 10 mg/ml protein extract and then use 1/3rd volume of this to 2/3rd volume of pyrene-actin (Cat. # AP05).  In this way there is a high enough concentration of protein to make a difference.  The extraction buffer should be 20 mM Hepes pH 7.5, 20 mM NaCl, plus any co-factors for your protein, and a protease inhibitor cocktail (e.g., Cat# PIC02).  Rinse the cells with an ice cold buffer and lyse cells with a 25 g bent over syringe needle or other device.  The control cell line is very critical because the actin polymerization reaction is very sensitive to slight differences in protein concentration or salts. 

If you have any questions concerning this product, please contact our Technical Service department at infohoelzel.de