STAT5B Antibody

What is STAT5B and what biological functions does it regulate?

STAT5B (Signal Transducer and Activator of Transcription 5B) is a transcription factor critically involved in regulating key biological pathways. It activates in response to various cytokines and growth factors, playing essential roles in cell development, proliferation, and survival mechanisms . STAT5B is particularly important in hematopoiesis, with significant functions in lymphocyte development, proliferation, and survival . The protein is approximately 90 kDa in molecular weight and is expressed in various cell types . Mutations in the STAT5B gene have been linked to impaired protein signaling that can manifest as stunted growth, autoimmunity, and immunodeficiency disorders, while hyperactivation of STAT5B is associated with blood malignancies and severe allergic inflammation . Understanding STAT5B's regulatory functions provides crucial insights into both normal cellular processes and disease mechanisms.

What types of STAT5B antibodies are available for research and how do they differ?

Several types of STAT5B antibodies are available for research applications, differing in their source, reactivity, and specific applications. Monoclonal antibodies like MAB1584 from R&D Systems are derived from single B-cell clones and offer high specificity, making them suitable for applications requiring consistent results across experiments . Polyclonal antibodies, typically raised in rabbits (as seen with antibody #34662 from Cell Signaling Technology), recognize multiple epitopes and may provide stronger signals in certain applications . Both types can be used for Western blotting and immunoprecipitation, though at different recommended dilutions (typically 1:1000 for Western blotting and 1:100 for immunoprecipitation with the CST antibody) . Species reactivity is an important consideration, with most commercial antibodies reacting with human, mouse, and rat STAT5B proteins . Phospho-specific antibodies (targeting Tyr705) are also available for studying STAT5B activation status . Researchers should select antibodies based on their specific experimental needs, the species they are working with, and the particular application they intend to use.

How can I validate the specificity of a STAT5B antibody for my research?

Validating STAT5B antibody specificity is crucial for ensuring reliable experimental results. A comprehensive validation approach should include multiple complementary methods. First, perform Western blot analysis using positive control cell lines with known STAT5B expression, such as HeLa, Daudi, or K562 human cell lines . The antibody should detect a band at the expected molecular weight of approximately 90 kDa . Include negative controls such as STAT5B knockout cells or siRNA-mediated knockdown samples to confirm specificity. To differentiate between STAT5A and STAT5B (which share high sequence homology), test the antibody against recombinant proteins of both types or use cells with selective expression of either protein. For phospho-specific STAT5B antibodies, validation should include treatment of cells with cytokines known to activate STAT5B signaling, followed by comparison to unstimulated controls . Cross-reactivity testing across multiple species should be performed if you plan to use the antibody in non-human models. Finally, consider immunoprecipitation followed by mass spectrometry as a definitive method to confirm that the antibody truly captures STAT5B protein. Document all validation steps methodically and include appropriate controls in each experiment to ensure reproducibility.

What are the optimal conditions for using STAT5B antibodies in Western blotting?

For optimal Western blot detection of STAT5B, proper sample preparation and protocol optimization are essential. Begin by lysing cells in an appropriate buffer such as SDT lysis buffer, loading approximately 6 μg of protein per sample as referenced in published protocols . The protein should be transferred to a PVDF membrane, which has been shown to work effectively with STAT5B antibodies . For blocking, 5% bovine serum albumin (BSA) in TBS-T for one hour at room temperature is recommended based on published methodologies . When using the Cell Signaling Technology STAT5B antibody (#34662), a dilution of 1:1000 has been validated for Western blotting . For R&D Systems' MAB1584, researchers have successfully used a concentration of 1 μg/mL . Incubate the membrane with primary antibody diluted in TBS-T containing 5% BSA overnight at 4°C . For detection, both HRP-conjugated secondary antibodies and infrared-labeled secondary antibodies (like anti-mouse IRDye 680 or anti-rabbit IRDye 800CW at 1:15,000 dilution) have been successfully employed . When studying phosphorylated STAT5B, phosphatase inhibitors must be included in all buffers during sample preparation, and phospho-specific antibodies targeting Tyr705 at a 1:500 dilution have been effectively used in published studies . Always include appropriate loading controls such as α-Tubulin to ensure equal protein loading across samples.

How should STAT5B antibodies be used in immunoprecipitation experiments?

For successful immunoprecipitation (IP) of STAT5B, several critical methodological considerations must be addressed. Based on validated protocols, begin with approximately 500-1000 μg of total protein from cell or tissue lysates prepared in a non-denaturing lysis buffer that preserves protein-protein interactions. The Cell Signaling Technology STAT5B antibody (#34662) has been validated for IP at a recommended dilution of 1:100 . Incubate the antibody with the lysate overnight at 4°C with gentle rotation to allow sufficient binding. For capturing the antibody-protein complex, both Protein A and Protein G sepharose/agarose beads can be used, though Protein A is often preferred for rabbit-derived antibodies . After incubation with the beads for 2-4 hours at 4°C, perform at least three stringent washes with cold wash buffer to remove non-specific interactions. Elute the immunoprecipitated proteins by boiling in SDS sample buffer for subsequent analysis by Western blotting. For confirmation of successful IP, probe the membrane with a different STAT5B antibody that recognizes a different epitope than the one used for IP, or use a direct detection method if the IP antibody is labeled. When studying STAT5B phosphorylation status, it's essential to maintain samples at 4°C throughout and include phosphatase inhibitors in all buffers. To investigate STAT5B interaction partners, consider performing co-immunoprecipitation experiments followed by mass spectrometry analysis to identify novel binding proteins in your specific experimental system.

What controls should be included when working with STAT5B antibodies?

Rigorous controls are essential when working with STAT5B antibodies to ensure experimental validity and reproducibility. Primary controls should include positive control samples from cell lines with confirmed STAT5B expression, such as HeLa, Daudi, or K562 human cell lines . Negative controls are equally important and should include STAT5B knockout or knockdown samples when possible, or at minimum, cell types known to express very low levels of STAT5B. Given the high homology between STAT5A and STAT5B, specificity controls to distinguish between these paralogs are critical - consider using recombinant STAT5A and STAT5B proteins or cells selectively expressing either protein. For phospho-specific experiments, include samples from cells treated with cytokines or growth factors known to activate STAT5B (positive control) alongside untreated samples (negative control) . Technical controls should include an isotype control antibody matching the STAT5B antibody's species and immunoglobulin class to identify non-specific binding. When performing Western blots, include a loading control (e.g., α-Tubulin) as demonstrated in published protocols . For immunoprecipitation experiments, include a "beads only" control (no antibody) to identify proteins that bind non-specifically to the matrix. Finally, antibody titration experiments should be performed to determine optimal concentration for each specific application, as excessive antibody can increase background while insufficient amounts may yield weak signals. Thorough documentation of all controls is essential for result interpretation and troubleshooting.

How can STAT5B antibodies be used to investigate STAT5B interaction partners?

Investigating STAT5B interaction partners provides crucial insights into its signaling networks and regulatory mechanisms. Co-immunoprecipitation (co-IP) using STAT5B antibodies represents the primary approach for studying protein-protein interactions. For this application, antibodies like Cell Signaling Technology's #34662 at a 1:100 dilution can effectively precipitate STAT5B along with its binding partners . When performing co-IP, use gentle lysis conditions to preserve native protein complexes, typically employing non-ionic detergents like NP-40 or Triton X-100 at low concentrations (0.1-0.5%). Following immunoprecipitation, Western blot analysis can detect specific suspected interaction partners, or mass spectrometry can identify novel binding proteins in an unbiased manner. Proximity ligation assays (PLA) offer an alternative approach, allowing visualization of protein interactions in situ by combining STAT5B antibodies with antibodies against potential interaction partners. For studying dynamic interactions, consider stimulating cells with cytokines or growth factors known to activate STAT5B signaling, then performing time-course co-IP experiments to capture temporal changes in the interactome. Bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET) approaches can also be employed, though these typically require expression of tagged proteins rather than relying on antibodies directly. When investigating interactions in the context of disease-associated mutations, researchers have found that STAT5B mutations like Y665F (located in the SH2 domain) can significantly alter dimerization and protein-protein interactions . Comparing the interactome of wild-type versus mutant STAT5B can reveal mechanistic insights into how these mutations contribute to pathological conditions, potentially identifying novel therapeutic targets.

What are common technical issues with STAT5B antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when working with STAT5B antibodies that require systematic troubleshooting approaches. One common issue is weak or absent signal in Western blotting, which may result from insufficient protein loading, antibody degradation, or suboptimal transfer. To address this, increase protein loading to 10-15 μg per lane, verify antibody integrity with positive controls such as HeLa or K562 cell lysates , and optimize transfer conditions for high molecular weight proteins (~90 kDa) . High background is another frequent problem, potentially caused by excessive antibody concentration or inadequate blocking. Dilute the antibody further (starting with 1:2000 for Western blotting instead of 1:1000) and extend blocking time to 2 hours with 5% BSA . Cross-reactivity between STAT5A and STAT5B can complicate interpretation due to their high sequence homology; validate antibody specificity using recombinant proteins or knockout samples, and consider using monoclonal antibodies that target unique epitopes. For phospho-specific antibodies, signal inconsistency often occurs due to rapid dephosphorylation; maintain samples at 4°C throughout processing and add phosphatase inhibitors to all buffers. In immunoprecipitation experiments, poor pull-down efficiency may result from suboptimal antibody-antigen binding conditions; try extending incubation time to overnight at 4°C and ensure gentle rotation . Non-specific bands in Western blots can be addressed by increasing washing stringency and duration after secondary antibody incubation. For detection issues, consider switching from chemiluminescence to more sensitive detection methods like infrared imaging systems, which have been successfully used with anti-mouse IRDye 680 and anti-rabbit IRDye 800CW secondary antibodies .

How can I optimize antibody dilutions for different experimental systems?

Optimizing antibody dilutions is crucial for achieving reliable and reproducible results across different experimental systems. Begin with a systematic titration approach, testing a range of dilutions based on manufacturer recommendations as starting points. For Western blotting with Cell Signaling Technology's STAT5B antibody #34662, the recommended 1:1000 dilution provides a useful starting point , while for R&D Systems' MAB1584, 1 μg/mL has been validated . Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) and test these simultaneously using identical samples to determine the optimal signal-to-noise ratio. For immunoprecipitation, a more concentrated antibody solution is typically required, with 1:100 dilution being recommended for CST's antibody #34662 . The optimal dilution can vary significantly depending on your specific experimental system, including cell type, protein expression level, and detection method. For high-expressing systems like HeLa, Daudi, or K562 cells , higher dilutions may be sufficient, while low-expressing systems may require more concentrated antibody solutions. When switching between different detection methods (e.g., from ECL to fluorescence-based detection), re-optimization is necessary as sensitivity differs considerably between systems. For phospho-specific STAT5B antibodies, published protocols have used 1:500 dilutions , but this may need adjustment based on activation status in your experimental conditions. Document all optimization experiments systematically, including images of blots at different exposures to support your selected dilution ratio. Finally, once an optimal dilution is established for your specific system, maintain consistency in antibody lot numbers when possible, as different lots may exhibit slight variations in affinity and specificity that could necessitate re-optimization.

How should researchers interpret contradictory results between different STAT5B antibodies?

Contradictory results between different STAT5B antibodies represent a significant challenge requiring careful analysis and methodological consideration. When faced with discrepancies, first examine the epitopes recognized by each antibody. Antibodies targeting different domains of STAT5B may yield different results depending on protein conformation, post-translational modifications, or the presence of interacting partners that might mask specific epitopes. Compare the validation data for each antibody, including Western blots demonstrating specificity across different cell lines like HeLa, Daudi, or K562 . Consider performing parallel experiments with multiple antibodies on the same samples under identical conditions to directly compare performance. If contradictions persist, evaluate each antibody's ability to distinguish between STAT5B and its paralog STAT5A, as cross-reactivity could explain discrepancies. For definitive validation, perform immunoprecipitation with one antibody followed by Western blotting with another, confirming they recognize the same protein. Additionally, knockdown or knockout experiments provide critical negative controls to establish specificity. When studying phosphorylated STAT5B, contradictions might arise from different antibodies' sensitivities to phosphorylation states; in this case, reference experiments demonstrating activation with cytokine treatment can help resolve inconsistencies . In the context of STAT5B mutations, certain antibodies might have altered affinity for mutant proteins; researchers studying Y665F and N642H mutations have observed differences in detection patterns that reflect functional consequences of these mutations rather than antibody limitations . Finally, when publishing research with contradictory results, transparently report all antibodies used, their sources, catalog numbers, and dilutions, along with detailed experimental conditions to allow proper scientific evaluation.

How are STAT5B antibodies being used in the study of hematological malignancies?

STAT5B antibodies have become instrumental in advancing our understanding of hematological malignancies through multiple sophisticated applications. In large granular lymphocyte (LGL) leukemia research, STAT5B antibodies have enabled the identification and characterization of somatic mutations, particularly the Y665F mutation in the SH2 domain and the N642H mutation, which are associated with distinct clinical presentations . Researchers employing both total STAT5B and phospho-specific antibodies (targeting Tyr705) have demonstrated that these mutations enhance STAT5B activation, with patients harboring the N642H mutation exhibiting more aggressive disease requiring therapeutic intervention . Western blot analysis using STAT5B antibodies allows researchers to quantify expression and activation levels across different patient samples, correlating these with clinical outcomes and treatment responses. Studies have successfully employed immunoprecipitation with STAT5B antibodies followed by mass spectrometry to identify novel interaction partners specific to malignant cells, potentially revealing new therapeutic targets. For investigating downstream effects of aberrant STAT5B activation, chromatin immunoprecipitation (ChIP) with STAT5B antibodies followed by sequencing has mapped altered genomic binding profiles in malignant versus normal cells. Flow cytometry with phospho-STAT5B antibodies enables researchers to identify specific subpopulations of malignant cells with activated STAT5B signaling within heterogeneous samples. In patient-derived xenograft models, immunohistochemistry with STAT5B antibodies helps track disease progression and response to targeted therapies. Additionally, recent research employing these antibodies has contributed to developing inhibitors targeting the STAT5B pathway, with Western blotting serving as a critical tool for assessing target engagement and efficacy in both preclinical models and patient samples .

What role do STAT5B antibodies play in studying immune disorders and autoimmunity?

STAT5B antibodies have become essential tools in investigating immune disorders and autoimmunity, enabling researchers to explore the molecular mechanisms underpinning these conditions. Given that mutations in the STAT5B gene are linked to impaired protein signaling associated with autoimmunity and immunodeficiency , these antibodies allow for precise examination of protein expression and activation status in patient samples. Western blot analysis using STAT5B antibodies at 1:1000 dilution enables quantification of total protein levels in different immune cell populations, while phospho-specific antibodies targeting Tyr705 at 1:500 dilution reveal activation patterns that may be dysregulated in disease states. Flow cytometry with fluorescently-labeled STAT5B antibodies permits single-cell analysis of expression and activation across various immune cell subsets, revealing cell type-specific abnormalities. In patients with STAT5B deficiency, immunoprecipitation followed by mass spectrometry has been employed to identify altered interaction partners that might contribute to pathogenesis. For investigating the functional consequences of STAT5B mutations, chromatin immunoprecipitation (ChIP) with STAT5B antibodies allows researchers to map changes in genomic binding sites and target gene expression. Immunohistochemistry using STAT5B antibodies on tissue samples from patients with autoimmune disorders helps visualize altered protein localization and expression patterns in affected tissues. Research has demonstrated that STAT5B plays a critical role in regulatory T cell development and function , making these antibodies valuable for studying immune tolerance breakdown in autoimmunity. Additionally, in models of severe allergic inflammation associated with STAT5B hyperactivation , these antibodies have helped elucidate the molecular pathways driving pathological immune responses, potentially identifying new therapeutic targets for intervention.

How can STAT5B antibodies contribute to developing targeted therapeutics?

STAT5B antibodies serve as critical tools in developing targeted therapeutics, facilitating multiple aspects of drug discovery and validation processes. In initial target validation stages, these antibodies enable precise quantification of STAT5B expression and activation across different disease models and patient samples, helping identify conditions where STAT5B-targeted therapies might be most effective. Western blotting with STAT5B antibodies at 1:1000 dilution and phospho-specific antibodies at 1:500 dilution allows researchers to measure target engagement of potential inhibitors, demonstrating whether compounds effectively reduce STAT5B phosphorylation and activation. High-throughput screening approaches often incorporate STAT5B antibodies in ELISA or AlphaLISA formats to rapidly evaluate large compound libraries for their ability to modulate STAT5B activity. For structure-based drug design, immunoprecipitation with STAT5B antibodies can isolate the protein for structural studies that inform rational drug development. In cellular models, immunofluorescence with STAT5B antibodies helps visualize inhibitor effects on STAT5B nuclear translocation, a critical step in its function as a transcription factor. When evaluating compounds targeting STAT5B mutations like Y665F or N642H found in certain malignancies , these antibodies enable assessment of mutation-specific effects, potentially leading to precision medicine approaches. In preclinical animal models, STAT5B antibodies facilitate pharmacodynamic studies, demonstrating whether drug candidates modulate the target as expected in vivo. Importantly, these antibodies also support biomarker development for clinical trials, establishing assays to measure STAT5B activation status in patient samples as indicators of treatment response. As therapeutic development progresses from bench to bedside, continuing refinement of antibody-based assays ensures consistent, reliable measurement of STAT5B modulation, supporting both regulatory approval processes and clinical implementation of targeted therapies.