UBASH3A Antibody

What is UBASH3A and why is it significant in immunological research?

UBASH3A (also known as STS-2, TULA, and CLIP4) is a protein primarily expressed in T cells that functions as a negative regulator of T-cell activation and function. It contains three functional domains: the N-terminal UBA (ubiquitin-associated), SH3 (SRC homology 3), and the COOH-terminal histidine phosphatase domain (also referred to as phosphoglycerate mutase-like [PGM]) . UBASH3A's significance stems from its association with multiple autoimmune diseases, as genetic variants in UBASH3A have been linked to at least five distinct autoimmune conditions, suggesting a broad role in autoimmunity regulation . Unlike its paralog UBASH3B, which is ubiquitously expressed, UBASH3A is specifically expressed in T cells and has been implicated in autoimmune disease susceptibility through genome-wide association studies . Mechanistically, UBASH3A attenuates NF-κB signal transduction upon T-cell receptor (TCR) stimulation by specifically suppressing the activation of the IκB kinase complex, representing a critical immunoregulatory pathway relevant to autoimmune disease development . Additionally, UBASH3A has been shown to regulate TCR-CD3 synthesis and turnover, further highlighting its multifaceted role in T-cell biology and making it an important target for immunological investigations .

What experimental approaches can be used to study UBASH3A expression in primary T cells?

Researchers can employ multiple techniques to investigate UBASH3A expression in primary T cells, with Western blotting being a fundamental approach. For optimal Western blot analysis, antibody dilutions between 1/500 and 1/3000 are recommended, though researchers should determine optimal concentrations through titration experiments . When designing experiments to measure UBASH3A expression, it is crucial to include appropriate loading controls such as GAPDH to accurately quantify relative protein abundance across different conditions or cell types . For investigating the relationship between UBASH3A expression and T-cell function, researchers should consider comparing IL-2 production in cells with different UBASH3A expression levels, as previous studies have demonstrated that modulating UBASH3A expression significantly impacts IL-2 production upon T-cell stimulation . When analyzing UBASH3A protein through Western blotting, researchers should be aware that both unmodified UBASH3A and its monoubiquitinated form may be detected, requiring careful interpretation of protein band patterns . For more sensitive detection of UBASH3A expression at the single-cell level, flow cytometry using fluorophore-conjugated UBASH3A antibodies can be employed following appropriate fixation and permeabilization protocols, enabling correlation with other cellular markers and functional readouts.

What are the key considerations when selecting a UBASH3A antibody for research applications?

When selecting a UBASH3A antibody, researchers should first consider the specific epitope targeted by the antibody and whether it corresponds to functionally relevant domains of UBASH3A. Antibodies targeting different domains (UBA, SH3, or PGM) may reveal distinct aspects of UBASH3A function . Researchers must verify reactivity and species specificity—for human studies, antibodies with confirmed human reactivity should be selected, as indicated in the product specifications . Application compatibility is another critical factor—researchers should choose antibodies validated for their intended applications, whether Western blotting, ELISA, immunoprecipitation, or other techniques . The clonality of the antibody impacts experimental outcomes; polyclonal antibodies offer broader epitope recognition but potential batch-to-batch variability, while monoclonal antibodies provide consistent specificity but may be limited to single epitopes . For quantitative studies, researchers should select antibodies with documented linear detection ranges and sensitivity appropriate for the expected expression levels of UBASH3A in their experimental system. For studies investigating interactions between UBASH3A and binding partners like TAK1, NEMO, or polyubiquitin chains, researchers should ensure the selected antibody does not interfere with these protein-protein interaction interfaces .

How can UBASH3A antibodies be optimized for studying NF-κB signaling pathways?

For studying UBASH3A's role in NF-κB signaling, researchers should implement co-immunoprecipitation protocols using anti-UBASH3A antibodies to capture and analyze protein complexes. The search results describe a method where 10 μg of anti-UBASH3A antibody is incubated with cell lysate overnight at 4°C, followed by addition of GammaBind Plus Sepharose beads for 2 hours at 4°C . This approach enables detection of novel interactions between UBASH3A and components of the NF-κB pathway. A series of stringent washes with different buffers (NETN buffer, followed by a buffer containing Tris-HCl, SDS, NP-40, and NaCl, then NETN again, and finally PBS) is crucial for reducing background and non-specific binding while maintaining specific interactions . Researchers should include appropriate negative controls, such as lysates from UBASH3A−/− cells processed identically to experimental samples, to distinguish between specific and non-specific signals . When eluting immunoprecipitated complexes, heating the beads for 10 minutes at 70°C in LDS buffer containing 50 mM DTT provides efficient recovery while maintaining protein complex integrity . Following immunoprecipitation, Western blotting for known NF-κB pathway components (particularly TAK1 and NEMO) will help elucidate UBASH3A's specific interactions in this signaling cascade . For functional studies, researchers can compare NF-κB activation markers in cells with different levels of UBASH3A expression (knockout, wild-type, and overexpression) following TCR stimulation to establish causality between UBASH3A activity and NF-κB signaling outcomes .

What methodological approaches can reveal UBASH3A's interactions with ubiquitin chains?

To investigate UBASH3A's interactions with ubiquitin chains, researchers should employ a pulldown assay using V5-tagged UBASH3A. This approach involves incubating whole-cell lysate from cells expressing V5-tagged UBASH3A with anti-V5 antibody for 4 hours at 4°C, followed by addition of Protein G Dynabeads and another hour of incubation . After washing the beads four times with cell lysis buffer, researchers should resuspend the beads in fresh lysis buffer and add specific types of ubiquitin oligomers (such as K48-linked) to investigate binding preferences . Western blotting can then be used to detect the interaction between UBASH3A and ubiquitin chains, with appropriate controls to ensure specificity of the observed interactions. For domain-specific analysis, researchers should generate UBASH3A constructs with mutations in key residues of the UBA domain, such as the K202R mutation described in the search results, which can be created using site-directed mutagenesis with specific primers (5'-gaccttggcccacaggttctacccccacc-3' and 5'-ggtgggggtagaacctgtgggccaaggtc-3') . Comparing the ubiquitin-binding capabilities of wild-type versus mutant UBASH3A provides insights into domain-specific functions. Mass spectrometry analysis of UBASH3A immunoprecipitates can further identify associated ubiquitinated proteins and characterize the types of ubiquitin chains involved in UBASH3A interactions, offering a comprehensive view of UBASH3A's role in ubiquitin-dependent signaling pathways .

How should researchers design experiments to investigate UBASH3A's effect on T-cell receptor dynamics?

To investigate UBASH3A's effect on TCR dynamics, researchers should utilize both UBASH3A knockout and overexpression systems in Jurkat T cells or primary T cells. The search results describe the generation of UBASH3A−/− clones via CRISPR/Cas9 editing and clones expressing V5-tagged UBASH3A, providing valuable experimental models . For measuring TCR-CD3 internalization rates, researchers should incubate cells with fluorescently labeled anti-CD3ε antibodies (such as APC-conjugated anti-CD3ε, clone HITa3) for 30 minutes on ice, wash them, and then transfer them to 37°C for varying time periods (0 to 60 minutes) . Surface-bound antibodies can be removed using an acidic buffer (100 mM glycine and 100 mM NaCl, pH 2.5) for 2 minutes at room temperature, allowing distinction between internalized and surface-bound receptors . Flow cytometry analysis of remaining fluorescence provides quantitative data on internalization rates across different UBASH3A expression conditions. Complementary experiments should assess total cellular levels of CD3 chains by Western blotting, with protein bands quantified using appropriate software and normalized to loading controls like GAPDH . The relative abundance of CD3 components can be calculated using the formula: (intensity of target protein in experimental sample / intensity of GAPDH in experimental sample) / (intensity of target protein in control / intensity of GAPDH in control) . For mechanistic insights, researchers should conduct similar experiments with UBASH3A mutants lacking specific domains or harboring point mutations that disrupt particular functions, such as the W279A mutation in the SH3 domain that affects protein-protein interactions .

What are the optimal conditions for Western blotting using UBASH3A antibodies?

For optimal Western blotting with UBASH3A antibodies, sample preparation should begin with complete cell lysis using buffers containing detergents and protease inhibitors to preserve protein integrity. The recommended antibody dilution range for Western blotting is 1/500 to 1/3000, though researchers should perform titration experiments to determine the optimal concentration for their specific experimental system . When detecting UBASH3A, researchers should be aware that both the unmodified protein and its monoubiquitinated form may appear as distinct bands, with the latter migrating at a higher molecular weight . For quantitative analysis, protein bands should be scanned using systems like the Typhoon 9200 laser scanner and quantified with software such as ImageQuant TL, with all target proteins normalized to loading controls such as GAPDH . The Western blotting protocol should include appropriate blocking steps (typically 5% non-fat milk or BSA in TBST) to minimize background signal and enhance specificity of antibody binding. For enhanced detection sensitivity, researchers can employ chemiluminescent substrates with extended signal duration or fluorescently labeled secondary antibodies that allow for multiplex detection and more accurate quantification. When comparing UBASH3A expression across different experimental conditions, technical replicates (minimum n=3) should be performed to ensure statistical validity of the observations . Researchers should also include positive controls (samples known to express UBASH3A) and negative controls (samples from UBASH3A knockout cells) to validate antibody specificity and establish detection thresholds .

What protocol should be followed for immunoprecipitation studies of UBASH3A and its interaction partners?

For immunoprecipitation studies investigating UBASH3A interactions, researchers should begin with careful preparation of cell lysates. The search results describe protocols using 500 μg of whole-cell lysate from cells expressing V5-tagged UBASH3A incubated with 0.5 μg of anti-V5 antibody for 4 hours at 4°C . Alternatively, endogenous UBASH3A can be immunoprecipitated using 10 μg of anti-UBASH3A antibody incubated with cell lysate overnight at 4°C . Following antibody incubation, researchers should add 20 μL of Protein G Dynabeads or 50 μL of GammaBind Plus Sepharose beads and continue incubation for 1-2 hours at 4°C to capture antibody-protein complexes . A stringent washing protocol is essential for reducing background and preserving specific interactions—the search results outline a comprehensive approach involving multiple washes with different buffers of varying stringency: NETN buffer (twice), a buffer containing Tris-HCl/SDS/NP-40/NaCl, NETN buffer again, and finally PBS (twice) . For elution of immunoprecipitated complexes, researchers should heat the beads for 10 minutes at 70°C in LDS buffer containing 50 mM DTT . When investigating specific interactions, co-immunoprecipitation followed by Western blotting for potential binding partners (such as TAK1, NEMO, or ubiquitin chains) provides direct evidence of protein-protein associations . Negative controls using lysates from UBASH3A knockout cells processed identically to experimental samples are essential for distinguishing between specific interactions and background binding . For investigating domain-specific interactions, researchers should utilize UBASH3A constructs with mutations in key residues, such as the W279A mutation in the SH3 domain, which can reveal the structural basis of specific protein-protein interactions .

How can researchers effectively use UBASH3A antibodies in flow cytometry applications?

For effective flow cytometry with UBASH3A antibodies, researchers must optimize fixation and permeabilization protocols to allow antibody access to intracellular UBASH3A while preserving cellular integrity and epitope recognition. Since UBASH3A is primarily expressed in T cells, researchers should incorporate T-cell markers (CD3, CD4, CD8) in their staining panels to correlate UBASH3A expression with specific T-cell subsets . When studying UBASH3A in relation to TCR dynamics, researchers can employ a protocol where cells are first labeled with fluorescently-conjugated anti-CD3ε antibody (such as APC-conjugated anti-CD3ε, clone HITa3) for surface staining, followed by fixation, permeabilization, and staining for intracellular UBASH3A . For analyzing TCR internalization, researchers should establish a time-course experiment where cells are labeled with anti-CD3ε antibody, incubated at 37°C for varying time periods (0-60 minutes), and then treated with an acidic buffer to remove surface-bound antibody, allowing measurement of internalized receptors by flow cytometry . Titration of UBASH3A antibody concentration is essential to determine the optimal signal-to-noise ratio, with appropriate isotype controls included to establish background staining levels. Compensation controls must be carefully prepared when using multiple fluorophores to correct for spectral overlap, ensuring accurate quantification of UBASH3A expression relative to other markers. For studies correlating UBASH3A expression with functional outcomes like IL-2 production, researchers can implement intracellular cytokine staining protocols in conjunction with UBASH3A staining following appropriate T-cell stimulation conditions . Single-color controls, fluorescence-minus-one (FMO) controls, and samples from UBASH3A knockout cells should be included to facilitate proper gating strategies and validate antibody specificity in flow cytometry applications .

How should researchers address inconsistent Western blot results with UBASH3A antibodies?

When encountering inconsistent Western blot results with UBASH3A antibodies, researchers should first examine sample preparation techniques. UBASH3A is subject to post-translational modifications, including monoubiquitination, which can affect its migration pattern on gels and result in multiple bands . Inconsistent signals may result from variable expression levels of UBASH3A across different cell types or activation states, as UBASH3A is primarily expressed in T cells and its expression may change upon T-cell activation . Researchers should ensure consistent protein loading through careful quantification of total protein concentration prior to gel loading and verification using housekeeping proteins such as GAPDH for normalization during analysis . The choice of lysis buffer can significantly impact the extraction efficiency of UBASH3A, particularly when investigating membrane-associated or nuclear fractions; comprehensive lysis buffers containing multiple detergents may improve consistency. Antibody specificity issues can be addressed by using lysates from UBASH3A knockout cells as negative controls to identify non-specific bands . If multiple antibodies targeting different epitopes of UBASH3A are available, comparing their staining patterns can help validate specific signals. Transfer efficiency problems may cause inconsistent results, particularly for proteins in certain molecular weight ranges; researchers should consider using stain-free gels or reversible protein stains to verify transfer efficiency before antibody incubation. For quantitative analysis, researchers should scan blots using systems like the Typhoon 9200 laser scanner and analyze band intensities with software such as ImageQuant TL, being careful to subtract background signals appropriately . Finally, researchers should always perform multiple biological and technical replicates (minimum n=3) and apply appropriate statistical analyses to determine whether observed variations represent biologically meaningful differences or technical artifacts .

What strategies can address epitope masking when studying UBASH3A in protein complexes?

When studying UBASH3A in protein complexes, epitope masking can occur when interaction partners occlude antibody binding sites. To address this challenge, researchers should consider using multiple antibodies targeting different epitopes across the UBASH3A protein. Since UBASH3A contains three distinct domains (UBA, SH3, and PGM), antibodies targeting different domains may provide complementary information about protein interactions . For co-immunoprecipitation studies, researchers might perform reciprocal immunoprecipitations, pulling down known interaction partners (such as TAK1 or NEMO) and blotting for UBASH3A, which may circumvent epitope masking that occurs when directly targeting UBASH3A . Pre-treatment of samples with crosslinking agents can stabilize transient protein-protein interactions but may further exacerbate epitope masking; in such cases, partial reversal of crosslinking prior to immunoblotting might improve epitope accessibility while maintaining complex integrity. When studying interactions with ubiquitin chains, researchers should be aware that the UBA domain of UBASH3A mediates these interactions, so antibodies targeting this region may show reduced binding in the presence of ubiquitinated proteins . Alternative detection strategies, such as mass spectrometry analysis of immunoprecipitated complexes, can provide complementary information about protein interactions without relying on epitope recognition by antibodies . For structural studies of UBASH3A complexes, mild detergents or alternative buffer conditions might help preserve interactions while improving epitope accessibility. Site-directed mutagenesis of key residues involved in protein-protein interactions, such as the W279A mutation in the SH3 domain, can generate valuable control samples where specific interactions are disrupted, helping distinguish between true interactions and non-specific associations .

How can researchers validate UBASH3A antibody specificity in knockout and overexpression systems?

To validate UBASH3A antibody specificity, researchers should implement a comprehensive approach comparing signals across three cell types: UBASH3A knockout cells, wild-type cells, and UBASH3A-overexpressing cells. The search results describe the generation of UBASH3A−/− clones via CRISPR/Cas9 editing and clones expressing V5-tagged UBASH3A, providing valuable experimental models for antibody validation . When validating antibodies by Western blotting, researchers should observe absence of specific bands in knockout samples, baseline expression in wild-type samples, and enhanced signal in overexpression samples, with band intensity correlating with the known expression levels in different overexpression clones (such as the varying expression observed in clones 1F6, 2F5, and 5E4) . For antibodies used in immunoprecipitation, researchers should confirm that immunoprecipitation from knockout cells yields no specific signal while demonstrating enrichment of UBASH3A from wild-type samples and further enrichment from overexpression samples . Flow cytometry validation should demonstrate absence of staining in knockout cells beyond isotype control levels, with positive staining in wild-type cells and enhanced signal in overexpression systems, allowing establishment of appropriate gating strategies based on these controls. When validating antibodies that recognize post-translational modifications of UBASH3A, such as its monoubiquitinated form, researchers should verify that both modified and unmodified forms are absent in knockout samples and present in expressing samples . For antibodies used in functional studies, researchers should confirm that the presence of the antibody does not interfere with the biological activities of UBASH3A, such as its ability to regulate IL-2 production or modulate TCR-CD3 expression and dynamics . Cross-reactivity with UBASH3B, the paralog of UBASH3A, should be assessed in systems where both proteins are expressed, with selective knockdown or knockout of each paralog to ensure antibody specificity .