Antibodies actin, preparation

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. 

Spudich and colleagues prepared bead-antibody-myosin complexes with varying amounts of myosin, HMM, and SHMM, and measured their speeds along Nitella actin fibers in the presence of ATP. The graph below sketches their results. 

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. 

Testis squashes can be stained with antibodies directed to specific proteins, with rho-damine-phalloidin to visualize F actin, and with DNA-binding dyes. Best results are obtained when these stainings are carried out in the following order immunostaining, F-actin staining, and DNA staining. This implies that multiply stained preparations are all mounted in the media described for DNA staining (see Protocol 5.8, step 2). 

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