This antibody is recommended for detection of actin in a broad spectrum of species such as human, mouse, rat, and bovine. The following protocols have been tested with this antibody:
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Background and Material
AAN02 is a mouse monoclonal antibody against actin protein. Actin is the major protein of the microfilament cytoskeletal system and is a key protein in various cell motility processes. The immunogen used for antibody production was purified actin protein from rabbit skeletal muscle. A characteristic actin band at 43 kDa is identified on Western blots (Fig. 1). The antibody has been shown to recognize α-skeletal, α-cardiac, α-smooth muscle, β-cytoplasmic, γ-smooth muscle and γ- cytoplasmic actin isotypes (Fig. 1) and has broad species cross-reactivity. AAN02 is purified by Protein G affinity chromatography and is supplied as a lyophilized white powder.
Figure 1. Western blot of purified actin and cell extracts probed with anti-actin antibody. Chemiluminescence detection of purified rabbit skeletal muscle actin (10 ng, lane 1), purified bovine cardiac muscle actin (10ng, lane 2), purified chicken gizzard muscle actin (10 ng, lane 3), purified non-muscle human platelet actin (10 ng, lane 4), platelet cell extract (5 µg, lane 5), platelet cell extract (5 µg, lane 6), and A431 cell extract (5 µg, lane 7). The actin band is indicated at 43 kDa. The blot was probed with a 1:1000 dilution of anti-actin antibody. 30 second exposure.
Figure 2. Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody. Swiss 3T3 cells were grown to 25% confluency on poly-lysine and laminin coverslips. 3T3 cells were fixed with PFA. Cells were permeabilized with methanol followed by 0.5% Triton X-100 as described in the method. Immunofluorescence staining using 1:500 dilution of anti-actin antibody in PBS is shown (green). The primary antibody was detected with a 1:500 dilution of goat anti-mouse alexfluor-488 conjugated antibody. DNA (blue) was stained with 50 nM DAPI in PBS. Image was taken with a 100X objective lens.
Figure 3. Comparison of fixation strategies used for Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody. Swiss 3T3 fibroblasts were plated on glass coverslips, grown to 30% confluency in DMEM plus 10% FBS and serum starved for 24 h in media containing 1% FBS followed by 24 h in serum free media. Cells were treated with a buffer control or 1 µg/ml of the Rho Activator (CN03) for 2 h at 37°C/5% CO2.to induce stress fibers. The 3T3 cells were then fixed with the following methods 1. PFA / phalloidin staining, 2. PFA-MeOH / AAN02 staining, 3. MeOH / AAN02 staining, 4. PFA / AAN02 staining. For condition 2 PFA fixation was followed by permeabilization with methanol as described in the method. Immunofluorescence staining using a 1:500 dilution of anti-actin antibody is shown (green). The primary antibody was detected with a 1:500 dilution of goat anti-mouse alexafluor-488 conjugated antibody. Images were taken with a 40X objective lens.
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