Human/Mouse STAT5b Antibody

Detection of Human, Mouse, and Rat STAT5b by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line, Daudi human Burkitt's lymphoma cell line, NIH-3T3 mouse embryonic fibroblast cell line, M1 mouse myeloid leukemia cell line, and PC-12 rat adrenal pheochromocytoma cell line. PVDF membrane was probed with 0.2 µg/mL of Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for STAT5b at approximately 90 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

STAT5b in HepG2 Human Cell Line. STAT5b was detected in immersion fixed HepG2 human hepatocellular carcinoma cell line using Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584) at 5 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; NL004) and counterstained with DAPI(blue). Specific staining was localized to cytoplasm and nucleus. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Immunoprecipitation of Human and Mouse STAT5b. HeLa human cervical epithelial carcinoma and M1 mouse myeloid leukemia cell line were untreated (-) or activated (+) with 100 ng/mL Recombinant Human IFN-gamma (285-IF) for 15 minutes. STAT5b was immunoprecipitated from lysates of 5 x 10⁶cells following incubation with 2 µg Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584) for 1 hour at room temperature. STAT5b-antibody complexes were absorbed using Protein A sepharose (Invitrogen, Catalog # 10-1041). Immunoprecipitated STAT5b was detected by Western blot using 1 µg/mL Human Phospho-STAT5a/b (Y699) Antigen Affinity-purified Polyclonal Antibody (AF4190). View our recommended buffer recipes for immunoprecipitation.

Detection of STAT5b in Jurkat Human Cell Line by Flow Cytometry. Jurkat human acute T cell leukemia cell line was stained with Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584, filled histogram) or control antibody (AB-105-C, open histogram), followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.

Detection of Human and Mouse STAT5b by Simple Western^TM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line and NIH‑3T3 mouse embryonic fibroblast cell line, loaded at 0.2 mg/mL. A specific band was detected for STAT5b at approximately 92 kDa (as indicated) using 10 µg/mL of Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

Western Blot Shows Human STAT5b Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and STAT5b knockout HeLa cell line (KO). PVDF membrane was probed with 0.2 µg/mL of Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for STAT5b at approximately 90 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Western Blot Shows Human STAT5b Specificity Using Knockout Cell Line. Western blot shows lysates of HAP1 human near-haploid cell line and STAT5b knockout HAP1 cell line (KO). Nitrocellulose membrane was probed with 0.2 µg/mL of Rabbit Anti-Human/Mouse STAT5b Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1584) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody. A specific band was detected for STAT5b at approximately 85 kDa (as indicated) in the parental HAP1 cell line, but is not detectable in knockout HAP1 cell line. The Ponceau stained transfer of the blot is shown. This experiment was conducted under reducing conditions. Image, protocol, and testing courtesy of YCharOS Inc. See ycharos.com for additional details.

Detection of Stat5b by Immunoprecipitation. Immunoprecipitation was performed on cell lysate of HAP1 human near-haploid cell line using 2.0 μg of Rabbit Anti-Human Stat5b Polyclonal Antibody (Catalog # AF1584) pre-coupled to protein G or protein A beads. Immunoprecipitated Stat5b was detected with a Mouse Anti-Stat5b antibody. The Ponceau stained transfers of each blot are shown. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitated. Image, protocol, and testing courtesy of YCharOS Inc. (ycharos.com).