STAT5A (Ab-780) Antibody

What is the specificity of STAT5A (Ab-780) Antibody and how was it generated?

The STAT5A (Ab-780) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of total STAT5A protein in human samples. It was produced by immunizing rabbits with a synthetic peptide corresponding to the sequence around amino acids 778-782 (R-L-S-P-P) of human STAT5A conjugated to KLH (Keyhole Limpet Hemocyanin). The antibody was purified through affinity chromatography using the epitope-specific peptide . This careful generation process ensures high specificity for STAT5A, with minimal cross-reactivity to other STAT family members.

What is the molecular basis for STAT5A function and why is position 780 significant?

STAT5A is a transcription factor that plays dual roles in signal transduction and transcriptional activation. It mediates cellular responses to cytokines and growth factors, including KITLG/SCF and ERBB4, and binds to GAS (Gamma-Activated Sequence) elements to activate prolactin-induced transcription . Position 780 is particularly significant as it contains a serine residue (S780) that undergoes phosphorylation. Research has demonstrated that S780 phosphorylation regulates STAT5A activity distinctly from the well-characterized tyrosine phosphorylation at Y694. S780 phosphorylation affects STAT5A's role in oncogenesis, particularly in luminal breast cancer, where it specifically influences the clonogenicity of cancer cells . The antibody's targeting of this region makes it valuable for studying this regulatory mechanism.

How should STAT5A (Ab-780) Antibody be stored to maintain optimal activity?

For optimal preservation of antibody activity, STAT5A (Ab-780) Antibody should be stored at -20°C for long-term preservation. For short-term use (up to 6 months), storage at 4°C is acceptable . The antibody is supplied at a concentration of 1.0 mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation helps maintain stability during storage. It is crucial to avoid repeated freeze-thaw cycles as they can degrade antibody performance . For laboratories conducting regular experiments with this antibody, preparing small working aliquots is recommended to minimize freeze-thaw cycles of the stock solution.

What are the validated applications for STAT5A (Ab-780) Antibody and their recommended protocols?

The STAT5A (Ab-780) Antibody has been validated for two primary applications:

Western Blotting (WB):

• Recommended dilution: 1:500 to 1:1000

• Expected molecular weight: 90 kDa

• Sample preparation: Total cell lysates or nuclear extracts

• Blocking recommendation: 5% non-fat milk in TBST

• Detection system: Standard HRP-conjugated secondary antibodies with ECL detection

Immunohistochemistry (IHC):

• Recommended dilution: 1:50 to 1:100

• Sample preparation: Formalin-fixed, paraffin-embedded sections

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Detection system: Polymer-based detection systems (e.g., HRP-polymer conjugates)

• Positive control: Human breast carcinoma tissue (as shown in validation images)

Validation data demonstrates successful detection of STAT5A in K562 cells by Western blot and in human breast carcinoma tissue by immunohistochemistry , confirming the antibody's specificity and performance in these applications.

How can researchers optimize detection of phosphorylated versus total STAT5A in experimental systems?

To effectively distinguish between phosphorylated and total STAT5A:

For Total STAT5A Detection:

• Use STAT5A (Ab-780) Antibody at recommended dilutions

• Include phosphatase inhibitors in lysis buffers to maintain the natural phosphorylation state

• Consider cell stimulation states based on experimental needs

For Phospho-Specific Detection:

• For pY694-STAT5: Use phospho-specific antibodies like the SRBCZX monoclonal antibody, which recognizes STAT5 phosphorylated at tyrosine 694

• For pS780-STAT5A: Use specific antibodies such as anti-STAT5a (phospho S780) antibody (ab30649)

• Use lambda phosphatase-treated samples as negative controls

Comparative Analysis Protocol:

• Prepare duplicate samples for parallel analysis

• Probe one membrane with STAT5A (Ab-780) Antibody for total STAT5A

• Probe another membrane with phospho-specific antibodies

• Normalize phospho-signal to total STAT5A signal for quantitative analysis

• Include appropriate positive controls (e.g., cytokine-stimulated cells)

This approach allows researchers to quantitatively assess the proportion of phosphorylated STAT5A relative to total STAT5A under various experimental conditions.

What cell types and experimental models are most appropriate for studying STAT5A using this antibody?

Based on research findings, the following cell types and models are particularly appropriate for STAT5A studies using this antibody:

Cell Lines:

• K562 cells: Used in validation Western blots, these BCR-ABL+ cells show strong STAT5A expression and activation

• MCF7 cells: Luminal breast cancer cells appropriate for studying STAT5A's role in breast cancer pathogenesis

• Jurkat cells: T-cell leukemia line showing STAT5A phosphorylation responses to stimuli

• 293T cells: Used in transfection studies for STAT5A overexpression and mutant analysis

Primary Cells:

• Hematopoietic cells: STAT5A plays critical roles in normal and malignant hematopoiesis

• Mammary epithelial cells: Important for studying STAT5A's role in lactation and breast cancer

Experimental Models:

• BCR-ABL-driven leukemia models: STAT5A is critical in the signaling network downstream of BCR-ABL

• Breast cancer models: Particularly useful for studying the differential roles of S726 and S780 phosphorylation

• Cytokine stimulation models: Using IL-2, IL-7, IL-15 (common gamma chain cytokines) or IL-3, IL-5, and GM-CSF (common beta chain cytokines) to activate STAT5 signaling

When designing experiments, researchers should consider that STAT5A and STAT5B have overlapping but distinct functions, particularly in hematopoietic cells, where STAT5B appears to be the dominant isoform downstream of BCR-ABL .

How can STAT5A (Ab-780) Antibody be used to investigate the distinct roles of STAT5A and STAT5B in hematopoiesis and leukemia?

The STAT5A (Ab-780) Antibody offers valuable capabilities for distinguishing STAT5A from STAT5B functions in hematopoiesis and leukemia research:

Experimental Approach:

• Comparative Expression Analysis:

  • Use STAT5A (Ab-780) Antibody alongside STAT5B-specific antibodies to quantify relative expression in various hematopoietic cell types

  • Analyze nuclear versus cytoplasmic fractions to assess differential translocation patterns

• Knockdown/Knockout Validation Studies:

  • In cells with STAT5A or STAT5B knockdown/knockout, use the antibody to confirm specificity and absence of compensatory changes

  • Combine with functional assays (proliferation, survival, differentiation) to attribute phenotypes to specific isoforms

• BCR-ABL Signaling Analysis:

  • Use in co-immunoprecipitation studies to identify STAT5A-specific interactors in BCR-ABL+ cells

  • Combine with phospho-specific antibodies to compare activation patterns between isoforms

Research Applications:

• Investigate the finding that STAT5B appears to be the dominant isoform downstream of BCR-ABL, facilitating transformation via suppression of IFN-α/β and IFN-γ signaling

• Explore how BCR-ABL directly activates STAT5B to a higher extent than STAT5A, as STAT5A remains partially cytoplasmic

• Study how imatinib-resistant cell lines upregulate STAT5A and become increasingly sensitive to tyrosine kinase inhibitors upon STAT5A knockdown

This approach can help elucidate the mechanistic basis for STAT5B's apparent predominance in BCR-ABL+ leukemias while clarifying the distinct contributions of STAT5A to disease progression and treatment response.

What is the significance of serine phosphorylation at position 780 of STAT5A in breast cancer pathogenesis?

Serine phosphorylation at position 780 of STAT5A represents a critical regulatory mechanism with distinct functional consequences in breast cancer:

Functional Impact of S780 Phosphorylation:

• Research has demonstrated that S780 phosphorylation specifically affects STAT5A-mediated clonogenicity in breast cancer

• MCF7 cells expressing S780A-STAT5A (preventing phosphorylation at this site) showed decreased colony formation in soft agar assays compared to wild-type STAT5A

• This effect differs from S726 phosphorylation, which primarily influences proliferation rather than clonogenicity

Experimental Protocol for Investigating S780 Function:

• Site-directed mutagenesis approach:

  • Generate STAT5A constructs with point mutations (S780A)

  • Create stable cell lines expressing these constructs in STAT5A-knockdown backgrounds

  • Perform RNA-sequencing to identify differentially regulated genes

  • Validate using functional assays (colony formation, proliferation, apoptosis)

• Phosphorylation-specific detection:

  • Use phospho-S780-specific antibodies to monitor this modification under various conditions

  • Compare with total STAT5A detection using STAT5A (Ab-780) Antibody

  • Correlate phosphorylation status with functional outcomes

Pathway Analysis Findings:
Research utilizing this approach revealed that loss of S780 phosphorylation significantly affects both prolactin-induced gene expression and functional pathways in breast cancer, including cell survival and colony formation. Ingenuity Pathway Analysis of RNA-seq data from cells expressing wild-type versus S780A-STAT5A identified distinct gene expression signatures and downstream pathways affected by this phosphorylation event .

This methodology demonstrates how researchers can utilize STAT5A (Ab-780) Antibody in combination with site-directed mutagenesis and phospho-specific antibodies to dissect the complex regulatory mechanisms governing STAT5A function in cancer.

How can STAT5A (Ab-780) Antibody be utilized in studying STAT5 inhibition as a potential therapeutic strategy?

STAT5A (Ab-780) Antibody can be instrumental in evaluating STAT5 inhibition strategies, which represent promising therapeutic approaches for STAT5-dependent malignancies:

Research Applications in Drug Development:

• Target Validation Studies:

  • Use the antibody to confirm STAT5A expression in patient-derived samples

  • Quantify nuclear versus cytoplasmic STAT5A to assess activation status

  • Correlate STAT5A levels with disease progression and treatment response

• Inhibitor Screening Protocols:

  • Western blot analysis with STAT5A (Ab-780) Antibody to evaluate:

    • Total STAT5A protein levels after inhibitor treatment

    • Changes in STAT5A subcellular localization

    • Potential degradation or post-translational modifications

  • Combine with phospho-specific antibodies to monitor inhibition of activation

• Mechanism of Action Studies:

  • Use in co-immunoprecipitation experiments to identify changes in STAT5A protein interactions

  • Chromatin immunoprecipitation (ChIP) assays to assess STAT5A binding to target genes

  • Immunofluorescence to visualize changes in STAT5A localization

Therapeutic Targeting Approaches:
Research has identified several STAT5 inhibition strategies that can be studied using this antibody:

The STAT5A (Ab-780) Antibody is particularly valuable for evaluating inhibitors that target the C-terminal region of STAT5A where the epitope is located, as these may affect detection if they alter antibody binding .

What are common technical challenges when using STAT5A (Ab-780) Antibody and how can they be addressed?

Researchers may encounter several technical challenges when working with STAT5A (Ab-780) Antibody, particularly in Western blotting and immunohistochemistry applications:

Western Blotting Challenges:

Immunohistochemistry Challenges:

General Optimization Strategies:

• For nuclear proteins like STAT5A, optimize nuclear extraction protocols

• In stimulation experiments, include time course analysis to capture optimal activation

• Incorporate peptide competition controls to confirm specificity

• When comparing STAT5A and STAT5B, run parallel blots with isoform-specific antibodies

These troubleshooting approaches are based on validation data showing successful detection of STAT5A in K562 cells by Western blot and in human breast carcinoma tissue by IHC .

How can researchers distinguish between STAT5A and STAT5B when using STAT5A (Ab-780) Antibody?

Distinguishing between the highly homologous STAT5A and STAT5B proteins requires careful experimental design:

Experimental Approaches for Isoform Discrimination:

• Antibody Selection Strategy:

  • STAT5A (Ab-780) Antibody targets a sequence around aa. 778-782 (R-L-S-P-P) in human STAT5A

  • This C-terminal region contains sequence differences between STAT5A and STAT5B

  • Use alongside STAT5B-specific antibodies targeting unique STAT5B epitopes

• Validation Methods:

  • Recombinant Protein Controls: Include purified recombinant STAT5A and STAT5B proteins as controls

  • Knockout/Knockdown Controls: Use STAT5A-/- and STAT5B-/- cell lines or knockdown samples

  • Band Migration Analysis: STAT5A (~94 kDa) and STAT5B (~92 kDa) can sometimes be distinguished by slight differences in migration on high-resolution gels

  • Immunoprecipitation-Western: Immunoprecipitate with isoform-specific antibodies, then blot with pan-STAT5 antibodies

• Cross-Reactivity Assessment Protocol:

  • Run Western blots with recombinant STAT proteins (STAT1-6)

  • Probe with STAT5A (Ab-780) Antibody

  • Compare with results from validated STAT5B-specific antibodies

  • Analyze band patterns for specificity (see validation Western blot data )

Practical Implementation:
Research suggests preferential use of this approach in models where one isoform predominates, such as BCR-ABL+ leukemia models where STAT5B plays a dominant role . When both isoforms are present, combining these discrimination approaches with functional studies (e.g., knockdown of individual isoforms followed by phenotypic analysis) provides the most comprehensive understanding of isoform-specific functions.

What controls should be included when using STAT5A (Ab-780) Antibody in experimental designs?

Rigorous experimental design requires appropriate controls to ensure valid interpretation of results when using STAT5A (Ab-780) Antibody:

Essential Controls for Western Blotting:

• Positive Controls:

  • K562 cell lysate (validated in product data sheets)

  • Cell lines with known STAT5A expression (MCF7, Jurkat)

  • Recombinant human STAT5A protein

• Negative Controls:

  • STAT5A-knockout or knockdown cell lysates

  • Cell lines with minimal STAT5A expression

  • Secondary antibody only (omit primary antibody)

• Specificity Controls:

  • Peptide competition assay using the immunizing peptide (R-L-S-P-P)

  • Non-specific peptide control to confirm specificity

• Loading Controls:

  • Housekeeping proteins (β-actin, GAPDH)

  • Total protein staining (Ponceau S, Coomassie)

Essential Controls for Immunohistochemistry:

• Tissue Controls:

  • Positive control: Human breast carcinoma tissue (validated in product data)

  • Negative control: Tissues known to lack STAT5A expression

  • STAT5A-knockout tissue (when available)

• Procedural Controls:

  • Omit primary antibody (secondary antibody only)

  • Isotype control (non-specific rabbit IgG at same concentration)

  • Peptide competition control (pre-incubation with immunizing peptide)

• Comparative Controls:

  • Serial sections stained with antibodies to related proteins (STAT5B)

  • Phospho-specific STAT5A antibodies on adjacent sections

Experimental Validation Controls:
For functional studies examining STAT5A phosphorylation and activity:

• Unstimulated versus cytokine-stimulated samples (e.g., prolactin stimulation)

• Tyrosine kinase inhibitor-treated samples (e.g., imatinib in BCR-ABL+ cells)

• Phosphatase-treated samples to eliminate phosphorylation

Inclusion of these comprehensive controls ensures reliable interpretation of experimental results and facilitates troubleshooting if unexpected results occur .

How does STAT5A function differ from STAT5B in BCR-ABL-driven leukemias and what methodologies can reveal these differences?

Despite their high homology, STAT5A and STAT5B exhibit distinct functions in BCR-ABL-driven leukemias that can be studied using specific methodological approaches:

Functional Differences:

• STAT5B, not STAT5A, is the dominant isoform downstream of BCR-ABL in leukemic transformation

• STAT5B facilitates transformation by suppressing IFN-α/β and IFN-γ signaling

• BCR-ABL directly activates STAT5B to a greater extent than STAT5A

• STAT5A remains partially cytoplasmic in BCR-ABL+ cells

• STAT5A knockdown in human cells shows minimal effect on survival, while STAT5B diminishment increases apoptosis and reduces self-renewal potential

• Paradoxically, imatinib-resistant cell lines upregulate STAT5A, and STAT5A knockdown increases sensitivity to tyrosine kinase inhibitors

Experimental Approaches to Study These Differences:

• Differential Localization Analysis:

  • Nuclear/cytoplasmic fractionation followed by Western blotting with STAT5A (Ab-780) Antibody

  • Immunofluorescence microscopy to visualize subcellular localization

  • Protocol: Compare nuclear/cytoplasmic ratios of STAT5A versus STAT5B in BCR-ABL+ cells before and after tyrosine kinase inhibitor treatment

• Isoform-Specific Knockdown Studies:

  • shRNA/siRNA targeting STAT5A or STAT5B specifically

  • Rescue experiments with wild-type or mutant constructs

  • Protocol: Assess apoptosis (cleaved caspase-3/7), proliferation, and colony formation after isoform-specific knockdown

• Transcriptomic Profiling:

  • RNA-seq after isoform-specific knockdown

  • ChIP-seq to identify distinct binding sites

  • Protocol: Compare gene expression profiles after STAT5A versus STAT5B knockdown in BCR-ABL+ cells to identify isoform-specific target genes

• Phosphorylation Dynamics:

  • Time-course analysis of tyrosine and serine phosphorylation

  • Phospho-specific antibodies in combination with STAT5A (Ab-780) Antibody

  • Protocol: Monitor kinetics of STAT5A and STAT5B phosphorylation after BCR-ABL activation or inhibition

These methodologies can reveal the mechanistic basis for STAT5B's privileged role in BCR-ABL+ leukemias while elucidating the context-specific contributions of STAT5A to disease progression and treatment response .

What unique insights can be gained by studying S780 phosphorylation of STAT5A in breast cancer research?

Phosphorylation of serine 780 in STAT5A represents a critical regulatory mechanism with specific functional consequences in breast cancer that can be studied using tailored experimental approaches:

Biological Significance of S780 Phosphorylation:

• S780 phosphorylation regulates STAT5A activity distinctly from the well-characterized Y694 phosphorylation

• In breast cancer, S780 and S726 phosphorylation have non-redundant roles, with S780 specifically affecting clonogenicity

• MCF7 cells expressing S780A-STAT5A (preventing phosphorylation) show decreased colony formation in soft agar assays

• RNA-sequencing analysis reveals that S780 phosphorylation regulates distinct gene expression patterns that influence cell survival and colony formation

Research Methodologies:

• Mutation-Based Functional Analysis:

  • Site-directed mutagenesis to generate S780A-STAT5A

  • Stable expression in STAT5A-knockdown backgrounds

  • Functional assays: soft agar colony formation, proliferation, apoptosis

  • Protocol: Compare phenotypes of wild-type versus S780A-STAT5A expressing cells in response to prolactin stimulation

• Phosphorylation Detection and Dynamics:

  • Use phospho-S780-specific antibodies alongside STAT5A (Ab-780) Antibody

  • Stimulation time courses with prolactin and other activators

  • In vitro kinase assays to identify responsible kinases

  • Protocol: Monitor S780 phosphorylation in response to various stimuli and correlate with functional outcomes

• Transcriptional Target Identification:

  • RNA-sequencing comparing wild-type versus S780A-STAT5A

  • ChIP-seq to identify differential genomic binding sites

  • Pathway analysis to identify functional consequences

  • Protocol: Perform Ingenuity Pathway Analysis on differentially expressed genes to predict functional effects

• Clinical Correlation Studies:

  • Immunohistochemistry on breast cancer tissue microarrays using phospho-S780 antibodies

  • Correlation with clinical parameters and outcomes

  • Protocol: Compare phospho-S780 levels across breast cancer subtypes and stages

This multi-faceted approach has revealed that S780 phosphorylation contributes to STAT5A's functional dichotomy in breast cancer, regulating both pro-differentiative and pro-proliferative target genes. The research demonstrates that S780 phosphorylation specifically affects clonogenicity, which differs from S726 phosphorylation that primarily influences proliferation .

How can researchers use STAT5A (Ab-780) Antibody to investigate potential STAT5-targeting therapeutic strategies?

STAT5A (Ab-780) Antibody provides researchers with valuable tools to evaluate promising therapeutic strategies targeting STAT5 in cancer and other diseases:

Therapeutic Targeting Approaches:

• Direct STAT5 Inhibition Strategies:

  • SH2 domain inhibitors (AC-4-130, IST5-002)

  • DNA-binding domain inhibitors

  • N-domain inhibitors affecting oligomerization

  • Selective STAT5B inhibitors (Capstafin, Stafib-1, Stafib-2)

• Indirect Approaches:

  • Upstream kinase inhibition (JAK inhibitors, TKIs)

  • Nuclear translocation inhibition

  • Protein stability modulators

  • Transcriptional or translational inhibition

Experimental Applications of STAT5A (Ab-780) Antibody:

• Target Validation and Expression Analysis:

  • Protocol: Quantify STAT5A expression across cell lines and patient samples using Western blotting and IHC

  • Application: Identify high STAT5A-expressing samples that may benefit from STAT5-targeted therapy

  • Methodology: Use STAT5A (Ab-780) Antibody at 1:500-1:1000 dilution for Western blot to screen cell lines and patient-derived xenograft models

• Compound Screening and Mechanism Validation:

  • Protocol: Treat cells with candidate compounds and analyze STAT5A expression, localization, and activity

  • Application: Identify compounds that effectively modulate STAT5A function

  • Methodology:

    • Western blotting with STAT5A (Ab-780) Antibody to assess total protein levels

    • Nuclear/cytoplasmic fractionation to evaluate nuclear translocation

    • Co-immunoprecipitation to examine protein-protein interactions

• Resistance Mechanism Studies:

  • Protocol: Compare STAT5A expression and localization in treatment-sensitive versus resistant cells

  • Application: Understand how STAT5A contributes to resistance to tyrosine kinase inhibitors

  • Methodology: Parallel analysis of STAT5A and STAT5B expression and phosphorylation in resistant models

• Combination Therapy Assessment:

  • Protocol: Treat cells with STAT5 inhibitors alone or in combination with other targeted agents

  • Application: Identify synergistic combinations for enhanced efficacy

  • Methodology: Western blotting with STAT5A (Ab-780) Antibody combined with viability and apoptosis assays

Research Findings on Potential Therapeutic Applications:
Studies have shown that disrupting the STAT5-BCR-ABL interaction could be therapeutically relevant in STAT5-dependent hematopoietic diseases. Inhibitors like IST5-002 block phosphorylation and nuclear translocation of STAT5A/B in BCR-ABL+ systems both in vitro and in vivo. Additionally, STAT5B-selective inhibitors (Stafib-1/2) target a STAT5B-specific amino acid in the linker domain, representing a novel design approach .