This is a knockout-validated antibody summary, based on the two publications "RKIP regulates MAP kinase signaling in cells with defective B-Raf activity" [1] and "Epidermal RAF prevents allergic skin disease" [2]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Western blot of Raf-B protein on the total lysates of primary keratinocyte isolated form wild type (WT) and B-Raf−/− mice from figure 6 [1].

Antibody: Raf-B, BRAF

Catalog number: sc-5284

Company: Santa Cruz

Summary: Mouse monoclonal IgG2a raised against amino acids 12-156 of Raf-B of human origin. Recommended for detection of Raf-B of mouse, rat and human origin by western blot, immunoprecipitation, immunofluorescence, immunohistochemistry (paraffin) and ELISA.

Western blot

Primary mouse keratinocytes were isolated from adult Braf KO mice, RIPA lysis buffer containing non-ionic (NP-40 or Triton X-100) and ionic (sodium dodecylsulfate and sodium deoxycholate) detergents (USB or Sigma) and the protease inhibitors (Calbiochem, Cat No: 539134).

Nitrocellulose membranes were blocked in Odyssey blocking buffer : 1X PBS (1:1) (Li-COR, Cat No: 927-40000)

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Figure 2. BRAF and RAF1 in wild-type and conditionally double knockout epidermal cells from 3 weeks old F/F2 and Δ/Δep2 (n = 4). ACTB is shown as a loading control, from figure 1 [2].

1:500 at 4°C with overnight.

Goat anti-Mouse IRdye 800CW (1: 8000)

detected and quantified using the Li-COR Odyssey system

Western blot

Epidermis was lysed with Laemmli buffer at 95°C for 5 minutes.

Lysates were immunoblotted at 1:1000 dilution.