In the following the protein preparation and purification procedures will be shortly illustrated. Then, the different buffer systems and the preparation the ac-tin medium will be explained. These procedures were conducted prior to the force and motility measurement in the actin in vitro medium.
Actin: Actin was purified from rabbit muscle according to the protocol by Spudich and Watt [107]. First 10 g of a powder-like acetone raw extract from the rabbit back and leg muscle is prepared. The powder was further extracted in Tris-Cl buffer (2mM Tris-Cl, 0.2 mM ATP, 0.5 mM β-mercaptoethanol, and 0.2 mMCaCl2, pH 8.0) and coarsely filtered. The supernatant fluid was then centrifuged at 10.000g for one hour to obtain a clear solution. From this solu-tion, the actin was allowed to polymerize by adding KCl to obtain 0.05 mM and MgCl2 to obtain 0.002mM in the solution. The following steps are identical to the actin cleaning procedure (centrifugation, pellet cleaning, and depolymeriza-tion see appendix 8.3).
Fluorescent actin was labeled with rhodamine-NHS (CAS Number 114616-32-9) according to [108]. Rhodamine-NHS binds to lysines in the actin mole-cule. Labeling the actin with rhodamine-NHS heavily interferes with its poly-merization properties. Therefore the degree of labeling was rather low, typically 0.8-1.2 lysines per actin molecule. The labeling reaction was performed in 50 mM PIPES buffer (pH 6.8), 50 mM KCl, 0.1 mM CaCl2, 0.2 mM ATP, 0.3 mM rhodamine-NHS and 40-60 µM F-actin. The degree of labeling was controlled by UV-VIS using the decrease of rhodamine-NHS absorbance at 567 nm, which occurs upon reaction with lysine. The reaction was stopped by adding excess of lysine if the desired degree of labeling was reached. The actin was then purified according to the protocol shown in appendix 8.3.
All g-actin solutions in were prepared and purified in M.-F. Carliers lab (LEBS-CNRS, 91198 Gif-sur-Yvette, France). The g-actin solution must not be frozen but kept on almost 0°C. Therefore, after shipping on ice, the g-actin solu-tion was always stored on ice in a refrigerator. The g-actin buffer is termed “g-buffer” and contains ions that prevent actin polymerization (see subsection
“buffers”). G-actin older than two month was either purified (see appendix 8.3) or replaced by freshly prepared actin.
4.1 Preparing the Actin In Vitro Medium
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Supplementary Proteins: Profilin was purified from bovine spleen [109].
Arp2/3 complex was purified from bovine brain [110] and for some experi-ments labeled with Alexa-488 maleimide. Human His-tagged N-WASP was ex-pressed in Sf9 cells using the baculovirus system [110]. All proteins were stored at -80°C in 5-10 µl aliquots. After melting, the protein aliquots were used for one week at maximum. All the supplementary proteins were also a gift from the M.-F. Carlier lab and shipped on dry ice (-78°C).
Buffers: The actin medium consists of rather complex buffer media. The medium in its final state is a mixture of tree buffer systems called “f-buffer”, “x-buffer” and “mix-“x-buffer”, see Table 4-1. The f-buffer is nothing but the stock solution of g-actin (in “g-buffer”) that has been supplemented with KCl and MgCl2 and EGTA. In the buffer, g-actin is allowed to polymerize to form f-actin. This is because of the combined effects of removing calcium ions with EGTA and adding Mg2+ and ATP [10]. The mix-buffer additionally adds an oxygen scavenger (DTT), photo bleaching inhibitor (DABCO) as well as ATP and Mg2+. The x-buffer is used to adjust the final concentration of actin and the supplementary proteins and also contains bovine serum albumin (BSA) to avoid non-specific protein interactions.
Since all actin assays start from a stock solution of actin in g-buffer, only the x-buffer and the mix-buffer have to be prepared prior to an experiment. Addi-tionally, a solution of KCl, MgCl and EGTA has to be prepared to transform the g-buffer into an f-buffer. The exact final composition depends on the amount of supplementary proteins that are added for completing the medium. ATP, Mg2+, DTT and DABCO concentrations may therefore vary, but this does not affect the actin polymerization. The composition of the buffers is shown in the table below.
g-buffer f-buffer x-buffer mix-buffer final
Table 4-1 Buffers composition and auxiliary components used for the actin poly-merization medium.
Some details on the buffer stability: In liquid condition, the mix-buffer can only be stored for three days, as it contains DTT and DABCO which are reac-tive and decaying in solution. ATP, DTT, and DABCO solutions in water can be stored at -20°C for several months. After re-melting the frozen aliquots of ATP, DTT and DABCO, the mix-buffer was prepared. The x-buffer solution is used for one week at maximum. After one week a fresh x-buffer solution was prepared.
Actin medium, final preparation: For the sake of simplicity, it is sufficient to work with a fixed total volume of the medium (usually 24-48 µl).1 This de-termines the amount of g-actin stock solution from which the final preparation
1 As the added amount of the protein stock solutions vary, the concentrations of e.g. ATP and MgCl2 also varies in the final mixture (see Table 4-1) but the effect on the network po-lymerization is negligible.