Immunofluorescence analysis of STAT5A [pY694] was done on 70% confluent log phase HeLa cells; untreated and treated (serum starved for 16 hours followed by treatment with 20 ng/ml IFN alpha for 1 hour). The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ABfinity™ STAT5A [pY694] Recombinant Rabbit Monoclonal Antibody (701063) at 2 µg-4 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing translocated STAT5A [pY694] in nucleus. Panel e shows competition with STAT5A [pY694] peptide. Panel f shows cytoplasmic localization of STAT5A [pY694] in untreated HeLa cells. The images were captured at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosphopeptide corresponding to amino acids 688–698 of human STAT5a|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||3µg|
|Flow Cytometry (Flow)||2-4µg/10^6 cells|
|Immunohistochemistry (Paraffin) (IHC (P))||1:20-1:200|
|Western Blot (WB)||2-5 ug/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
STAT5, consisting of isoform a and b, belongs to the family of STAT (signal transducers and activators of transcription) proteins which are phosphorylated by receptor associated kinases, translocate to the nucleus, and act as transcription factors in response to cytokines and growth factors. STAT5a is activated by IL2, IL3, IL7, GM-CSF, erythropoietin, thrombopoietin, and various growth hormones. In humans, the STAT5a gene is located on chromosome 17.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.