Description: This SRBCZX monoclonal antibody recognizes signal transducer and activator of transcription 5 (STAT5) when phosphorylated on tyrosine 694. STAT proteins are activated by ligand binding to receptors, such as cytokine receptors, that associate with Janus kinase (JAK) family members. Following their phosphorylation by JAKs, STAT proteins translocate to the nucleus where they bind to DNA and regulate transcription of specific genes in a cell type- and cytokine-specific manner. In response to cytokines that signal through the common gamma chain such as IL-2, IL-7, and IL-15, STAT5 is phosphorylated on tyrosine 694 by JAK1 and JAK3. Cytokines such as IL-3, IL-5, and GM-CSF that signal via the common beta chain induce STAT5 phosphorylation on tyrosine 694 by JAK 2. Phosphorylation of STAT5 on tyrosine 694 is essential for STAT5 dimer formation, nuclear translocation, and DNA binding activity.
Specificity of this SRBCZX clone was determined by ELISA and flow cytometry.
Applications Reported: This SRBCZX antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested: This SRBCZX antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 µL (0.06 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.
Staining Protocol: We recommend using Protocol C: Two-step protocol: Fixation/Methanol. Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins and Protocol B: One-step protocol: intracellular (nuclear) proteins cannot be used. All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online.
PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.
Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.
Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.
Filtration: 0.2 µm post-manufacturing filtered.
STAT5 alpha is a member of the STAT family of transcription factors. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. STAT5 alpha is activated by, and mediates the responses of many cell ligands, such as IL2, IL3, IL7 GM-CSF, erythropoietin, thrombopoietin, and different growth hormones. Activation of STAT5 alpha in myeloma and lymphoma associated with a TEL/JAK2 gene fusion is independent of cell stimulus and has been shown to be essential for the tumorigenesis. The mouse counterpart of STAT5 alpha is found to induce the expression of BCL2L1/BCL-X(L), which suggests the antiapoptotic function of this protein in cells. STAT5 alpha, along with STAT5 beta, were identified in mouse. The amino acid sequences of STAT5 alpha and STAT5 beta show 96% sequence similarity, and both proteins are co expressed in most tissues in virgin and lactating mice. However, differential accumulation of STAT5 alpha and STAT5 beta mRNA has been reported for both muscle and mammary tissue. STAT5 alpha is critically involved in a variety of physiological functions, including reproduction, lactation, immune function and somatic growth.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Protein Aliases: Mammary gland factor; mammary gland factor STAT5A; Signal transducer and activator of transcription 5A; STAT; STAT5B
Gene Aliases: AA959963; MGF; Mpf; STAT5; STAT5A
Molecular Function: DNA-binding transcription factor