Drugs and Buffers

Actin stability and polymerization is highly dependent on buffer conditions, therefore it is vital to use the right buffers. For example, actin polymerization is dependent on the concentration of magnesium chloride which should be in the range of 100 µM to 5 mM (see the "About actin" pages for more information). Cytoskeleton provides a basic set of tris buffers to allow the researcher to consistently work with monomer or polymer actin (see list below).

 In addition, we provide a range of fluorescent phalloidins which can be used to stain F-actin structures in cells as well as stabilize F-actin filaments for in vitro use, including microscopy (see PHDG1, PHDH1PHDN1, PHDR1 datasheets below for examples). 

Cytoskeleton's actin products have been cited hundreds of times over the past two decades. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.

 

Phalloidin (rhodamine): 14uM (Cat. # PHDR1)

Ezratty, E. J., Partridge, M. A. and Gundersen, G. G. (2005). Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat. Cell Biol. 7, 581-590.
Gomes, E. R., Jani, S. and Gundersen, G. G. (2005). Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121, 451-463.

Question 1:  What buffers do I need for an actin polymerization assay? 

Answer 1: Cytoskeleton offers all of the necessary reagents and buffers for performing actin polymerization assays in a convenient kit (Cat. # BK003) with a detailed manual.  The kit contains pyrene-labeled muscle actin (Cat. # AP05), general actin buffer (Cat. # BSA01-010), polymerization buffer (Cat. # BSA02-001), ATP stock (Cat. # BSA04), Tris-HCl pH 7.5 and a 96 well plate. 

To polymerize actin, three buffers are recommended.  The first buffer is a General Actin Buffer (Cat. # BSA01-010) that is comprised of 5 mM Tris-HCl pH 8.0 and 0.2 mM CaCl2.  On the day of use, this buffer is supplemented with fresh ATP to a final concentration of 0.2 mM (Cat. # BSA04-001).  The second buffer is an Actin Polymerization Buffer (Cat. # BSA02-001) which consists of 50 mM KCl, 2 mM MgCl2, and 1 mM ATP(added fresh) at 1X strength.  We sell these reagents and the polymerization buffer is sold at 10X strength.   The third buffer is 100 mM Tris-HCl, pH 7.5 for resuspending stock ATP (Cat. # BSA04). 

 

Question 2:  What buffers are included in the G-actin/F-actin In Vivo Assay Kit? 

Answer 2: Cytoskeleton offers all of the necessary reagents and buffers for measuring G-actin and F-actin levels in cell or tissue extracts in a convenient kit (Cat. # BK037).  The G-actin/F-actin In Vivo Assay Kit (Cat. # BK037) includes a Lysis and F-actin stabilization buffer (50 mM PIPES pH 6.9, 50 mM KCl, 5 mM MgCl2, 5 mM EGTA, 5% (v/v) Glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% 2-mercapto-ethanol and 0.001% Antifoam C), 100X protease inhibitor cocktail (Cat. # PIC02; 0.4 mM Tosyl arginine methyl ester, 1.5 mM leupeptin, 1 mM pepstatin A, 1 M benzamidine), ATP (Cat. # BSA04-001), F-actin enhancing solution (phalloidin), F-actin depolymerization solution (urea), G-actin control standard (Cat. # AKL99-A), G-actin antibody (Cat. # AAN01), 5X SDS sample buffer and anhydrous DMSO.

 

For more infomation, please contact tservice@cytoskeleton.com with questions regarding these products.