|Detection of Human and Mouse Oct‑3/4 by Western Blot. Western blot shows lysates of BG01V human embryonic stem cells and D3 mouse embryonic stem cell line. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Oct‑3/4 at approximately 48 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
Chromatin Immunoprecipitation (ChIP)
|Detection of Oct‑3/4-regulated Genes by Chromatin Immunoprecipitation. BG01V human embryonic stem cells were fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Oct‑3/4/DNA complexes were immunoprecipitated using 5 μg Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) or control antibody (Catalog # AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (Catalog # BAF109). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The nanog promoter was detected by standard PCR.|
|Alkaline Phosphatase and Oct‑3/4 in BG01V Human Stem Cells. Alkaline phosphatase (ALPL) and Oct‑3/4 were detected in human BG01V embryonic stem cells using 10 µg/mL Human/Mouse/Rat ALPL Monoclonal Antibody (Catalog # MAB1448) and 10 µg/mL Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759). Cells were incubated with primary antibodies for 3 hours at room temperature. Cells were stained for ALPL using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (pseudo-stained green; Catalog # NL007), and stained for Oct-3/4 using the NorthernLights 637-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL002). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
|Detection of Human Oct‑3/4 by Simple WesternTM. Simple Western lane view shows lysates of BG01V human embryonic stem cells and NTera‑2 human testicular embryonic carcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for Oct‑3/4 at approximately 50 kDa (as indicated) using 20 µg/mL of Goat Anti-Human/Mouse Oct‑3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.|
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Immunocytochemistry/Immunofluorescence: Human/Mouse Oct-3/4 Antibody [AF1759]