The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 10 µg/ml.
Use a concentration of 0.5 µg/ml. Predicted molecular weight: 42 kDa.
Use at 2 µg/mg of lysate. Use Protein G.
1/50. Boil tissue sections in 10mM citrate buffer, pH 6.0 for 10 - 20 minutes followed by cooling at room temperature for 20 minutes.
FunctionActins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells.
Involvement in diseaseDefects in ACTA1 are the cause of nemaline myopathy type 3 (NEM3) [MIM:161800]. A form of nemaline myopathy. Nemaline myopathies are muscular disorders characterized by muscle weakness of varying severity and onset, and abnormal thread-or rod-like structures in muscle fibers on histologic examination. The phenotype at histological level is variable. Some patients present areas devoid of oxidative activity containg (cores) within myofibers. Core lesions are unstructured and poorly circumscribed. Defects in ACTA1 are a cause of myopathy congenital with excess of thin myofilaments (MPCETM) [MIM:161800]. A congenital muscular disorder characterized at histological level by areas of sarcoplasm devoid of normal myofibrils and mitochondria, and replaced with dense masses of thin filaments. Central cores, rods, ragged red fibers, and necrosis are absent. Defects in ACTA1 are a cause of congenital myopathy with fiber-type disproportion (CFTD) [MIM:255310]; also known as congenital fiber-type disproportion myopathy (CFTDM). CFTD is a genetically heterogeneous disorder in which there is relative hypotrophy of type 1 muscle fibers compared to type 2 fibers on skeletal muscle biopsy. However, these findings are not specific and can be found in many different myopathic and neuropathic conditions.
Immunocytochemistry/ Immunofluorescence - Anti-Actin antibody [ACTN05; same as C4] - BSA and Azide free (ab76548)
ab76548 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab76548 at 10µg/ml overnight at +4°C. The secondary antibody (green) was a goat anti-mouse DyLight® 488 (ab96879) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in methanol fixed (100%, 5min) Hek293, HepG2, and MCF-7 cells at 10ug/ml.
Western blot - Actin antibody [ACTN05; same as C4] - BSA and Azide free (ab76548)
Anti-Actin antibody [ACTN05; same as C4] (ab76548) at 0.5 µg/ml + Human cells 8
Predicted band size : 42 kDa Observed band size : 42 kDa
Immunoprecipitation - Actin antibody [ACTN05; same as C4] - BSA and Azide free (ab76548)
Actin was immmunoprecipitated from human cells 8 using ab76548, followed by Western blotting using ab76548 at 0.5 µg/ml.
Flow Cytometry-Anti-Actin antibody [ACTN05; same as C4] - BSA and Azide free(ab76548)
Overlay histogram showing HeLa cells stained with ab76548 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76548, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
References for Anti-Actin antibody [ACTN05; same as C4] (ab76548)
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