STATs were originally identified as two novel DNA-binding proteins (STAT1 and STAT2) which were found to play essential roles in interferon IFNa-and IFNg-regulated gene expression.2 Following the identification of these first two family members, five additional mammalian STAT proteins were cloned and characterized including: STAT3, STAT4, STAT5a, STAT5b, and STAT6 (1,2). All STAT proteins share several conserved structural and functional domains(1,2): (1) conserved amino terminal domain (2) DNA binding domain (3) SH3-like region (4) conserved SH2 domain responsible for: recruitment to the receptor, interaction with JAKs, STAT dimerization. (5) conserved tyrosine residue (Y) whose phosphorylation is required for dimerization and DNA binding (6) carboxy terminal activation domain. In unstimulated cells, STAT proteins exist largely in the cytoplasm as latent transcription factors. In response to treatment of target cells with cytokines or in some cases growth factors, specific STATs undergo tyrosine phosphorylation, homo-or heterodimerization, nuclear translocation, and DNA binding resulting in the transcriptional activation of distinct target genes(1,2). At least one, and oftentimes several STAT proteins are activated in response to all cytokines which utilize cytokine receptor superfamily members. Nevertheless, a striking specificity of STAT activation is seen in response to individual cytokines (1). STAT5 or MGF (mammary growth factor) was originally isolated by purification of tyrosine phosphorylated DNA binding proteins from prolactin stimulated mammary tissue and from IL-3 stimulated myeloid cells(1,2,3,4). Subsequently, two highly related, but distinct Stat5 genes (Stat5a and Stat5b) were identified in mouse(5,6). The amino acid sequences of STAT5a and STAT 5b show ~ 96% sequence similarity, and both proteins are co-expressed in most tissues of both virgin and lactating mice(5,7,10). However, differential accumulation of STAT5a and STAT5b mRNAs have been reported for both muscle and mammary tissue. STAT5 has been reported to be activated by multiple cytokines including, IL-2, IL-7, IL-9, IL-15, IL-3, IL-5, IL-6, GM-CSF, TPO, EPO, GH, and Prolactin (PRL)(1–10). Both the STAT5a and STAT5b proteins recognize the GAS (gamma interferon activating sequence) site TTCNNNGAA, and the preferred DNA-binding consensus site has been identified as TTCC(A>T)GGAA(1,2). Western blot analysis of the full-length STAT5a protein, isolated from a variety of cultured cells, indicates that the protein migrates with a molecular weight of ~95kD. Carboxy-terminal truncated forms of the STAT5a protein have also been reported(6,8).

Suitable for use in FLISA, Western Blot, Immunoprecipitation. Other applications not tested.

FLISA: 0.1–1ug/ml Immunoprecipitation: 5ug Western Blot: 1–3ug/ml Gel Mobility Shift Assay: 2.5–5ug

Optimal dilutions to be determined by the researcher.

Store product at 4°C in the dark. DO NOT FREEZE! Stable at 4°C for 12 months after receipt as an undiluted liquid. Dilute required amount only prior to immediate use. Further dilutions can be made in assay buffer. Caution: MaxLight™650 conjugates are sensitive to light. For maximum recovery of product, centrifuge the original vial prior to removing the cap.

Note: Applications are based on unconjugated antibody.