Immunostaining Antibody Validation Protocol

Anti-BrdU antibody (mouse monoclonal B44) was purchased from BD Biosciences. anti-GFP antibody (A11122) purchased from Molecular Probes. Antibodies against acetylated α-tubulin (mouse monoclonal 6-11B-1, T6793) and γ-tubulin (rabbit polyclonal, T5192) were purchased from Sigma-Aldrich. Rabbit anti-IFT88 (13967-1-AP), TULP3 (13637-1-AP), and Arl13b (17711-1-AP) were purchased from Proteintech. Mouse anti-GAPDH antibody (GT239) was purchased from GeneTex. Rabbit antiserum against Human ZNF423 (amino acid residues 247–407 relative to reference sequence NP_055884.2), was prepared and affinity-purified against the immunogen [32 (link)]. Commercial antibodies against ZNF423 (Santa Cruz Biotechnology sc-48785, aa 1–105; Sigma-Aldrich SAB2104426, aa 1234–1283) showed similar pattern (L. Flores-Garcia and B.A.H.). Western blots were developed with infrared-conjugated secondary antibodies (Rockland), detected on a Li-Cor Odyssey Imaging Station, and quantified in the ImageJ software package.

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Hong C.J, & Hamilton B.A. (2016). Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function. PLoS Genetics, 12(10), e1006357.

Publication 2016

anti-GFP antibody (A11122) Arl13b (17711-1-AP) infrared-conjugated secondary antibodies Odyssey Imaging Station

Amino acid Anti antibody Antibodies Antiserum Brdu Gapdh Human Immunogen Molecular probes Mouse Rabbit Tubulin Western blots α tubulin

Corresponding Organization : University of California, San Diego

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of various cellular components (e.g., BrdU, GFP, acetylated α-tubulin, γ-tubulin, IFT88, TULP3, Arl13b, GAPDH, ZNF423)

control variables

None explicitly mentioned

controls

Positive controls: None specified
Negative controls: None specified

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