Invitrogen™ Phospho-STAT1 (Tyr701) Monoclonal Antibody (KIKSI0803), eFluor™ 660, eBioscience™, Invitrogen™

Catalog No.	Quantity
50-112-4478	100 Tests

Catalog No. 50-112-4478 Supplier Invitrogen™ Supplier No. 50900842

Description

Mouse Monoclonal Antibody

Description: This KIKSI0803 monoclonal antibody recognizes signal transducer and activator of transcription 1 (STAT1) when phosphorylated on tyrosine 701. 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. Phosphorylation of STAT1 on tyrosine 701 by JAK1 and JAK2 is essential for STAT1 dimer formation, nuclear translocation, and DNA binding activity. In response to IFN gamma stimulation, STAT1 homodimerizes or forms heterodimers with STAT3 that can bind to GAS (IFN gamma-activated sequence) promoter elements. In response to either IFN alpha or IFN beta stimulation, STAT1 forms a heterodimer with STAT2 that can bind ISRE (IFN-stimulated response element) promoter elements. Specificity of this KIKSI0803 clone was determined by ELISA, flow cytometry, and western blotting. Applications Reported: This KIKSI0803 antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This KIKSI0803 antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 μL (0.06 μg) per test.

STAT1 (signal transducers and activators of transcription 1) is a member of the STAT family of transcription factors. STAT1 can be activated by interferon-alpha, interferon-gamma, EGF, PDGF and IL6. STAT1 is known to regulate several genes which are involved in cell growth, apoptosis, immune responses, and lipid metabolism. Further, STAT1 plays an important role in mediating cell viability in response to different cell stimuli and pathogen exposure. The STAT1 gene is located on chromosome 2. STAT1 is activated to regulate gene expression in response to extracellular signaling polypeptides including cytokines, interferons, and growth factors. After phosphorylation by JAK tyrosine kinases, STAT1 enters the nucleus to regulate transcription of many different genes. Among the seven STATs types, STAT1, STAT3, STAT5a, and STAT5b have a wide activation profile. STAT1 is activated by many different ligands including IFN family (IFN-Alpha, IFN-Beta, IFN-gamma and IL-10), gp130 family (IL-6, IL-11, LIF, CNTF, and G-CSF), and receptor tyrosine kinases (EGF, PDGF, and CSF-1). Two alternatively spliced transcript variants encoding distinct isoforms of STAT1 have been described.

Specifications

• Antigen: Phospho-STAT1 (Tyr701)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: KIKSI0803
• Concentration: 5 μL/Test
• Conjugate: eFluor 660
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: STAT1
• Gene Accession No.: P42224
• Gene Alias: 2010005J02Rik; AA408197; CANDF7; DD6G4-4; DKFZp686B04100; IMD31A; IMD31B; IMD31C; ISGF-3; LOW QUALITY PROTEIN: signal transducer and activator of transcription 1-alpha/beta; OTTHUMP00000165046; OTTHUMP00000205845; signal transducer and activator of transcription 1; signal transducer and activator of transcription 1, 91kD; signal transducer and activator of transcription 1, 91kDa; signal transducer and activator of transcription 1-alpha/beta; signal transducer and activator of transcription-1; STAT; STAT 1; STAT1; STAT-1; stat1 alpha; STAT91; transcription factor ISGF-3; transcription factor ISGF-3 components p91/p84
• Gene Symbols: STAT1
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 6772
• Target Species: Human
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG1 κ

Frequently Asked Questions (FAQs)

Can the eFluor Organic fluorochromes be used for intracellular staining?

Yes, the eFluor Organic fluorochromes can be used for intracellular staining. The eFluor organic fluorochromes maintain bright signal and require minimal changes in compensation when fixed with eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) and Permeabilization Buffer (Cat. No. 00-8333-56) or 1-step Fix/Lyse Solution (Cat. No. 00-5333-54, 00-5333-57) (as compared to live cells).

How does eBioscience determine the specificity of their phosphorylation-specific antibodies?

Antibodies are tested and validated in a variety of ways. First, we verify that in a panel of cells in which different pathways have been induced, we see phosphorylation specific staining only in the cells in which the specific pathway of interest has been activated. Second, we verify that phosphorylation specific staining is observed only in cell types in which the protein is expressed and not in cell types in which the protein is not expressed. Third, whenever possible, western immunoblotting is used to confirm the presence of a band(s) of the appropriate size(s) in stimulated/treated cells (and not in unstimulated/untreated cells, as appropriate).

Is the eFluor 660 fluorochrome compatible with Anti-Cy5/Alexa Fluor 647 beads?

Yes, in-house studies have demonstrated that the eFluor 660 fluorochrome is recognized by Anti-Cy5/Alexa Fluor 647 beads. Side by side studies with Alexa Fluor 647 versus eFluor 660 conjugated antibodies have demonstrated comparable results.

What is the difference between eFluor Organic Dyes and eVolve Qdots?

The eFluor Organic Dyes (eFluor 450, APC-eFluor 780, PerCP-eFluor 710, eFluor 710) are conventional fluorochromes. In contrast, the eVolve line of products are Quantum dots.

How long can cells that have been fixed and placed in methanol be stored for use with eBioscience antibodies? What is the recommended storage temperature? How about storage of fixed cells in eBioscience fixation buffers?

We have data demonstrating that fixed cells stored in methanol are stable at -20 degrees C or at -80 degrees C for several weeks. We do not recommend storing fixed cells in eBioscience Foxp3/Transcription Factor Buffer. However, cells fixed with eBioscience Foxp3/Transcription Factor Buffer or eBioscience Intracellular (IC) fixation buffer can be stored in eBioscience IC fixation buffer for up to 3 days at 4 degrees C in the dark.
Note: Please be aware that higher compensation values may be seen with tandem dyes if any other fixative is used apart from eBioscience IC fixation buffer, for 3-day storage.

What is the optimal protocol for eBioscience phosphorylation-specific antibodies?

Each antibody has been tested in three different buffer systems: IC fixation and permeabilization, Foxp3/Transcription Factor Buffer, and IC fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for the specific antibody.

What is the best way to maintain surface staining when using the methanol-based protocol?

Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor 450, FITC, and eFluor 660).
Please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. We recommend staining after stimulation/treatment, fixation, and methanol treatment.

Can I use other buffer systems from other companies when using eBioscience phosphorylation-specific antibodies?

In general, you can use other companies' buffer systems provided that their buffers are of a similar composition as the buffers recommended. Be aware that results will vary depending upon the buffer system used. eBioscience antibodies have been optimized for use in eBioscience buffers and we have not tested all of our antibodies in other buffer systems. Thus, we highly recommend using our optimized protocol and buffer systems.

What are the appropriate controls to run when using eBioscience phosphorylation-specific antibodies?

It is important to understand whether the stimulation/treatment results in an upregulation or a down regulation of the phosphorylation event. Thus, it is recommended that changes in phosphorylation levels in stimulated/treated versus unstimulated/untreated cells be compared. A histogram or density plot overlay these two treatments will provide a better assessment of a change in phosphorylation event than using isotype controls.

Can I perform phosphorylation specific staining and stain for intracellular cytokines at the same time when using eBioscience phosphorylation-specific antibodies?

In theory, if the buffer systems are compatible for the antibodies of interest, this can be done. In practice, cytokine translation and phosphorylation events do not typically occur in the same timeframe, therefore, it may be difficult to detect both events in the same sample. The results will be depended on the kinetics and should be determined for each system and protein of interest.

When using eBioscience phosphorylation-specific antibodies, can I perform phosphorylation specific staining together with transcription factor staining at the same time? If so, what buffer and protocol do I use?

In theory, this can be done if the antibodies against both the transcription factor and the phosphorylated protein work in the same buffer system. For example, Anti-Human/Mouse phsopho-H2AX and all transcription factor eBioscience antibodies offered will work in the Foxp3/Transcription Factor Buffer System (Cat. No. 00-5523-00).

For eBioscience phosphorylation-specific antibodies, can total and phosphorylated protein be analyzed in parallel?

In theory, this can be done if the antibodies to both proteins work in the same buffer system and if the antibodies recognize different epitopes. Please refer to the individual Technical Data Sheets for information about buffer compatibility. Also, consider using eBioscience InstantOne ELISA Kits for immunoassay quantitation of both total and phosphorylated proteins such as AKT1/2/3, JNK1/2/3, NFκB, STAT3, and STAT5.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

For Research Use Only.