Actin preparation methods

Simon, J.R., and Taylor, D.L. (1988) Preparation of a fluorescent analog Acetamidofluoresceinyl labeled dictyostelium discoideum a-actin. In Methods in Enzymology, (R.B. Vallee, ed.), Vol. 134, p. 47. Academic Press, San Diego. 

Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

Mommaerts (1961a) asserts that less than 50% of the protein in actin solutions prepared by Straub s method polymerizes to F-actin on addition of salt. Correcting for this, the L-myosin-actin ratio for maximum viscosity would be 3 1. The purity of actin preparations can also be tested, however, by making use of the ability to combine with myosin, the impurities remaining in solution when the actomyosin complex is precipitated (A. Weber, 1949). By this test, our actin preparations are 60-90 % pure, and impurities were allowed for in the ratios reported by Jaisle. 

For these experiments, they used a more well-defined method for attaching the myosin to the beads. The beads were clumps of killed bacterial (Staphylococcus aureus) cells. These cells have a protein on their surface that binds to the Fc region of antibody molecules (Fig. 5-2la). The antibodies, in turn, bind to several (unknown) places along the tail of the myosin molecule. When bead-antibody-myosin complexes were prepared with intact myosin molecules, they would move along Nitella actin fibers in the presence of ATP. 

The full biological activity of C2 toxin, a mixture of components I and II, is obtained by activation of the toxin with trypsin (Miyake and Ohi-shi, 1987 Ohishi et al., 1980 Ohishi et al., 1980 Ohishi, 1987). As described in Section 9.2 (Assay method for the toxin), the full activity of the toxin is produced by a mixture of untrypsinized component I and trypsinized component II. This indicates that activation of the toxin is brought about by the molecular cleavage of component II, but not of component I, by trypsin. Therefore, to study the biological activity of botulinum C2 toxin and the effect of ADP-ribosylation of cytoplasmic actin by C2 toxin on whole cells, it is essential to prepare activated component II (trypsinized component II). 

Quantification of the amount of toxin-ADP-ribosylated actin in the intact cell can be done by the method of differential ADP-ribosylation. However, pP]ADP-ribosylation of cell lysates prepared by standard procedures is critical because C2I can still work on ice. Thus, under the conditions of short term intoxication or analysis of the time course of intoxication, C2I continues to ADP-ribosylate actin during the preparation of the lysates. This results in an apparently higher amount of actin modified in the intact cell. To avoid this artefact, it is recommended that lysis of cells should be performed in the presence of p2p]NAD (as given in 11.2.3.2 ). 

This protein was recently prepared from cod muscles and investigated by Connell (1954). Acetone-dried muscle fiber was extracted with water by the method of Feuer, et al. (1948) omitting the final treatment with Na2COa (Tsao and Bailey, 1953). The G-actin obtained polymerizes on addition of salts to a viscous solution of F-actin showing pronounced double refrac-... 

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

From the observations described here and other reports in the literature (Fowler and Bennett, 1984 Broschat and Burgess, 1986 Heald and Hitchcock-DeGregori, 1988), there does not appear to be a direct cause-and-effect relationship between propensity for head-to-tail polymerizahon and affinity of actin binding. There may be several reasons for this, including a less than satisfactory experimental method for measuring head-to-taU interaction. Viscosity measurements can only be considered a qualitative tool for this purpose and are subject to major uncertainties in their experimental application. Clearly a more reliable experimental method is needed. The methodology by which the TM is prepared may also have significant effects on the polymerization properties of the protein. Methionines, of which there are three in residues... 

When actin and myosin have once combined to give actomyosin, it is not possible by any known method to separate them completely on a preparative scale. There is no doubt, however, that natural actomyosin is really a complex of actin and mj osin, (a) because Straub (1942) obtained in small yield from actomyosin the same actin as obtained from the dry acetone powder of muscle, and (b) natural and artificial actomyosins react with ATP in the same typical manner (Section III, 5d). It can therefore be concluded that complex formation is thermodynamically irreversible, for by repeated fractional precipitation a preparation can be obtained from muscle extracts in which no free L-myosin can be detected by methods at present available. The ultracentrifugal peak of L-myosin reappears, however, when the actomyosin in solution by its history and its properties, e.g., disappearance of ATP-sensitivity, may be regarded as denatured (Portzehl et al., 1950 see also Johnson and Landolt, 1950). 

Actin was prepared from rabbit skeletal muscle by the method of Spudich and Watt [19] and was further pinified with gel-filtration over Sephadex G-150 gel [20]. Monomeric actin, labeled with the fluorophore tetramethylrhodamine maleimide, was a generous gift of Dr Ewa Prochniewcz at the Department of Biochemistry, University of Minnesota. It was concluded from a preliminary experiment that Spudich-Watt actin contained minor factorfs) which greatly accelerated the polymerization of actin at 0.5 mM Ca and at 8°C upon the addition of Ca ions, 100 pM Spudich-Watt actin rapidly polymerized, whereas the purified actin did not. 

Cortese et al. [10] demonstrated that the vesicles assumed irregular shapes and polymerized actin filaments exhibited a heterogeneous distribution within the vesicles, which is quite different from our results. The difference in the method of preparation of actin-encapsulating vesicles (diethyl ether injection method versus swelling method) or of the cation species and the concentration may be the source. The latter may have significantly affected the polymerization rate and hence the final vesicle shape. 

Finally, the issue of firontend purification for the collected test material is perhaps the most important aspect of sample preparation. Currently there are various methods that utilize standard separation techniques to remove most abundant proteins prior to MS, i.e., removal of some 20 high-abundance proteins and better visualization of low-abundance proteins (Sigma-Aldrich kits). However, in most cases the removal of these proteins may in fact compromise the detection of biomarkers or their partners, since in many instances, viral infection leads to overexpression of the most abundant proteins such as the actins, keratins, tubulins, cyclophilins, vimentin, and HSPs among others. Therefore, future attempts at the identification of biomarkers would have to define not only the most high- and low-abundance proteins but also their partners and possible modifications. 

The chain of reactions involved in muscle contraction, from the signal at the neuro-muscular junction and the release of calcium by the sarcoplasmic reticulum, to the force development by the contractile apparatus, is currently studied by a wide variety of techniques. These involve experiments with preparations at every level of organization from pure protdns in solution to intact muscle. Some of the important new developments of methods, which can be used for the study of the same reaction step in solution and within cells, have used the contractile system as a paradigm. The application of pressure relaxation to the study of actin-miyosin interaction in solution as well as of tension changes in fibres is discussed in section 6.4. The initiation of reactions by the photochemical release of ATP from inactive precursors, discussed in section 8.4, has provided data for the rates of steps of myosin ATPase in contracting muscle fibres. Many dilferent signals can be used for the kinetic resolution of steps involved in... 

This invention relates to methods for preparing photo-patterned mono- or polychromatic, polarizing films. The polarizer can be pixelated into a number of small regions wherein some of the regions have one orientation of the principal neutral or color absorbing axis and some other of the said regions have another orientation of the principal neutral or color absorbing axis. The axis orientation is determined by the polarization vector of actinic radiation and the multi-axes orientation is possible by a separated masked exposure. This polarizer can be placed on the interior substrate surface of the LCD cell. 

---