γ-Actin antibody | knockout validation | Sigma-Aldrich A8481

This is a knockout-validated antibody summary, based on the publication "A genome editing vector that enables easy selection and identification of knockout cells", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

γ-Actin antibody | knockout validation | Sigma-Aldrich A8481 figure 1
Figure 1. (A) Western blotting analysis of each knockout cell line using an antibody specific for each actin isoform. The lane indicated as Cont was loaded with whole cell lysate of the parental cell line, U2OS. Beta-tubulin was used as the internal control for Western blotting. (B) Immunofluorescence of knockout cell lines. Cells were stained with the same isoform-specific antibodies as those used in Western blotting. Pseudocolor is not consistent with the fluorescent dye color of the secondary antibodies. All images are shown with the same magnification. Bar: 100 um. Please see Figure 4 in the article [1].
Antibody information

Mouse monoclonal IgG1

Company: Sigma-Aldrich

Antibody: γ-Actin

Catalog number: A8481

Summary: Mouse monoclonal IgG1 against purified bovine brain cytoplasmic γ-Actin and the KLH-conjugated amino terminal peptide of human γ-Actin (amino acids 2-15). Reacts with human, bovine, canine, mouse, hamster and chicken. Suitable for western blot, immunofluorescence, immunohistochemistry and ELISA.

Validation Method

Western blot | Immunocytochemistry

Sample

Parental and γ-Actin-KO U2OS cells.

Primary incubation

WB: 1:2,000 dilution.

Secondary incubation

WB: 1:3,000 dilution horseradish peroxidase–conjugated anti-mouse antibody (KPL, Gaithersburg, MD).

IC: anti-mouse IgG1 conjugated with Alexa Fluor 488 (Invitrogen).

Detection

WB: the chemiluminescence signal (Western BLoT Hyper HRP Substrate, Takara-Bio) was detected with a C-DiGit Blot Scanner (LiCor, Lincoln, NE).

IC: conventional fluorescence microscope (IX-81, Olympus, Tokyo, Japan) equipped with an Orca Flash2.8 C-MOS camera (Hamamatsu Photonics, Hamamatsu, Japan).

References
  1. Nagasaki A, Kato Y, Meguro K, Yamagishi A, Nakamura C, Uyeda T. A genome editing vector that enables easy selection and identification of knockout cells. Plasmid. 2018;98:37-44 pubmed publisher