This is a knockout-validated antibody summary, based on two publications "Conditional mutations of beta-catenin and APC reveal roles for canonical Wnt signaling in lens differentiation" [2] and "Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells" [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

[enlarge]

Figure 1. E14.5 wild-type; mouse embryo eye section stained with anti β-catenin (Transduction Labs #610154; 150dpi image 7x5cm) showing β-catenin reactivity (green; Alexa488, Gt-anti mouse IgG) along cell membranes. Objective magnification 20x. Courtesy of the authors.

Mouse monoclonal IgG1

Company: Transduction Labs, now part of BD Biosciences

Antibody: beta-catenin

Catalog number: 610154

Summary: Reactive against human, mouse, rat, dog, chicken. Can be used in Western blot, immunohistochemistry, immunoprecipitation, immunofluorescence. The immunogen is mouse β-catenin aa. 571-781.

[enlarge]

Figure 2. E14.5 conditional KO of Ctnnb1; mouse embryo eye section stained with anti β-catenin (Transduction Labs #610154; 150dpi image 7x5cm) showing loss of staining in the lens only. Ctnnb1 CKO as per Cain et al., 2008; Dev Biol, 321:420-433. Objective magnification 20x. Courtesy of the authors.

Immunohistochemistry

Mouse eye lenses from wild-type and mutant βCATEx310 at E14.5.

3% to 5% normal goat serum (NGS) in 0.1% BSA/PBS for 20 minutes.

1:100 diluted in blocking solution at 4°C overnight.

AlexaFluor488-conjugated anti-mouse IgG (1:500; A-11001; Invitrogen) diluted in blocking solution for 2 hours at room temperature.

[enlarge]

Figure 3. Western blotting experiments confirm complete lack of CTNNB1 protein expression in biallelically targeted H9 hESCs colonies. GAPDH is used as loading control. From [1].

Western blot | Immunocytochemistry

[enlarge]

Figure 4. Confocal images show CTNNB1 and Nanog expression in wild-type (WT) and CTNNB1 knockout (KO) H9 hESCs by immunostaining. Scale bar, 50 mm. From [1].

WT and CTNNB1 paired-KO H9 hESCs. WB: Cells were lysed in RIPA+ buffer. IF: Coverslip cultures were fixed in 4% paraformaldehyde at room temperature for 10 min.

IF: 10% donkey serum, 0.1% Triton X-100 in PBS for 1 hr .

WB: 1:1,000 dilution.

IF: 1:1,000 dilution at 4°C overnight.

WB: Secondary antibodies were purchased from Jackson ImmunoResearch.

IF: Fluorescently conjugated secondary antibodies (1:1,000; Jackson Laboratories) for 1 hr.

If the antibody described in this summary is a polyclonal antibody, since polyclonal antibodies are of limited quantity, please inquire the supplier whether any current polyclonal antibody with the same catalog number is exactly the same as the one described in this summary. Sometimes, different bleeds or different animals are used, usually with a different lot number. In such cases, the result in this summary may not apply to the new antibody with the same catalog number.