STUB1 Antibody, Biotin conjugated

What is STUB1 and what cellular functions does it regulate?

STUB1 is a 34.5 kDa co-chaperone protein encoded by the STUB1 gene that functions as an E3 ubiquitin ligase. It is highly conserved across species and expressed in most tissues . STUB1 plays a crucial role in protein quality control by targeting misfolded proteins for proteasomal degradation through its ubiquitin ligase activity. Recent studies have identified STUB1 as a critical regulator of immune signaling, particularly through destabilization of the IFNγ receptor complex .

STUB1 contains three key functional domains: an N-terminal tetratricopeptide repeat (TPR) domain that mediates interactions with chaperone proteins like HSP70, a central coiled-coil domain, and a C-terminal U-box domain responsible for its E3 ubiquitin ligase activity. Through these domains, STUB1 regulates protein stability, cellular stress responses, and immune signaling pathways .

How does a biotin-conjugated STUB1 antibody compare to unconjugated versions for experimental applications?

Biotin-conjugated STUB1 antibodies offer distinct advantages over unconjugated versions, particularly for certain detection methods. The biotin conjugation provides signal amplification through high-affinity interaction with streptavidin-coupled detection systems, enhancing sensitivity in techniques like immunohistochemistry, ELISA, and flow cytometry.

For optimal experimental outcomes, researchers should consider the following differences:

Methodologically, when using biotin-conjugated antibodies, researchers should include an endogenous biotin blocking step when working with biotin-rich tissues (e.g., liver, kidney) to minimize background signal.

What are the validated species reactivity patterns for STUB1 antibodies?

Commercial STUB1 antibodies have been validated across multiple species, with confirmed reactivity in human, mouse, and rat samples . Specifically, Western blot analysis has demonstrated positive detection in various human cell lines (A549, HEK-293, HeLa, MCF-7), mouse cell lines (NIH/3T3), rat cell lines (PC-12), as well as tissue samples including mouse brain, liver, and kidney, and rat kidney .

For immunohistochemistry applications, STUB1 antibodies have shown positive detection in human colon cancer tissue, human liver tissue, human heart tissue, human skeletal muscle tissue, and mouse skeletal muscle tissue . This broad cross-species reactivity reflects the highly conserved nature of STUB1 protein across mammalian species.

What are the optimal sample preparation and dilution protocols for different applications of STUB1 antibodies?

Proper sample preparation and antibody dilution are critical for successful STUB1 detection across different experimental techniques. Based on validated protocols, the following methodological approaches are recommended:

For Western Blot (WB):

• Prepare cell or tissue lysates in RIPA buffer containing protease inhibitors

• Load 20-40 μg of total protein per lane

• Use recommended dilutions between 1:500-1:6000, with optimal ranges of 1:1000-1:2000 for unconjugated antibodies

• For biotin-conjugated antibodies, use streptavidin-HRP at 1:5000-1:20000 dilution

• Include positive control samples (HEK-293 or HeLa cell lysates)

For Immunohistochemistry (IHC):

• Fix tissues in 10% neutral buffered formalin

• Perform antigen retrieval with TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Use antibody dilutions between 1:50-1:500, with optimal range around 1:100-1:200

• For biotin-conjugated antibodies, include a biotin blocking step

• Use streptavidin-HRP detection system

For Immunofluorescence (IF):

• Fix cells in 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100

• Block with 5% normal serum

• Use antibody dilutions between 1:50-1:200

• For biotin-conjugated antibodies, use fluorophore-conjugated streptavidin for detection

How should researchers validate STUB1 knockout or knockdown models?

Validation of STUB1 knockout or knockdown models requires a multi-faceted approach to confirm specific targeting and rule out off-target effects. Based on published research methodologies, the following systematic validation approach is recommended:

• Genomic validation:

  • For CRISPR/Cas9 knockout: Sequence the targeted locus to confirm insertion/deletion

  • For siRNA/shRNA: Verify specificity of targeting sequence using BLAST analysis

• Transcript level validation:

  • Perform RT-qPCR to quantify STUB1 mRNA levels

  • Include primers spanning different exons to detect any alternatively spliced variants

• Protein level validation:

  • Western blot using antibodies targeting different epitopes of STUB1

  • Compare results from multiple antibodies to confirm complete protein loss

• Functional validation:

  • Assess levels of known STUB1 substrates (IFNγ-R1, JAK1) by Western blot

  • Measure proteasomal degradation of these substrates using proteasome inhibitors like MG132

  • Evaluate downstream signaling pathways (e.g., IFNγ signaling) known to be regulated by STUB1

• Rescue experiments:

  • Reintroduce wildtype STUB1 to confirm phenotype reversibility

  • Include domain mutants (TPR-deficient, UBOX-deficient, E3 ligase dead H260Q) to assess domain-specific functions

This comprehensive validation approach ensures that observed phenotypes are specifically attributable to STUB1 modulation.

What controls are essential when using STUB1 antibodies for studies of the IFNγ receptor complex?

When investigating STUB1's role in regulating the IFNγ receptor complex, several critical controls must be incorporated to ensure experimental validity:

• Antibody specificity controls:

  • Include STUB1 knockout/knockdown samples as negative controls

  • Use recombinant STUB1 protein as a positive control for antibody validation

  • Compare results from multiple STUB1 antibodies targeting different epitopes

• Protein interaction controls:

  • Include TPR domain mutants that cannot interact with HSP70 chaperones

  • Test K30 mutants with altered binding capacity to JAK1

  • Use UBOX domain mutants to distinguish between binding and E3 ligase functions

• Proteasomal degradation controls:

  • Include MG132 treatment to block proteasomal degradation

  • Compare effects in wildtype versus STUB1-deficient cells

  • Monitor both IFNγ-R1 and JAK1 levels simultaneously

• Signaling pathway controls:

  • Include IFNγ stimulation time course

  • Monitor phosphorylation of downstream STAT proteins

  • Include JAK inhibitors to confirm pathway specificity

• Domain-specific function controls:

  • Compare effects of wildtype STUB1 versus E3 ligase dead mutant (H260Q)

  • Use TPR domain-deficient and UBOX domain-deficient variants

  • Perform reconstitution experiments in STUB1-deficient cells

These controls help distinguish between specific STUB1-mediated effects and potential artifacts or indirect consequences of experimental manipulation.

What are common sources of variability in STUB1 antibody detection and how can they be addressed?

Researchers often encounter variability in STUB1 detection across different experimental conditions. Understanding these sources of variability and implementing appropriate strategies can improve data consistency and reliability:

• Antibody lot-to-lot variation:

  • Perform validation testing when switching to a new antibody lot

  • Maintain reference samples for comparison across experiments

  • Consider pooling antibody lots for long-term projects

• Sample preparation inconsistencies:

  • Standardize tissue or cell lysis protocols

  • Monitor protein degradation using additional housekeeping proteins

  • Prepare fresh lysates when possible, as STUB1 can be subject to degradation during storage

• Post-translational modifications affecting epitope recognition:

  • STUB1 undergoes auto-ubiquitination that may mask antibody epitopes

  • Include deubiquitinating enzyme treatment in parallel samples

  • Compare results using antibodies targeting different STUB1 regions

• Proteasome activity differences between samples:

  • Consider pre-treating samples with proteasome inhibitors to normalize STUB1 substrate levels

  • Monitor proteasome activity across experimental conditions

  • Include MG132 treatment as a control condition

• Cross-reactivity with related proteins:

  • Verify antibody specificity using STUB1 knockout samples

  • Perform immunoprecipitation followed by mass spectrometry to identify potential cross-reactive proteins

  • Use multiple antibodies targeting different epitopes to confirm findings

By systematically addressing these variables, researchers can achieve more consistent and interpretable results when studying STUB1 biology.

How can researchers resolve discrepancies between protein levels detected by different methods?

When discrepancies arise between STUB1 levels detected by different methods (e.g., Western blot versus immunofluorescence), a systematic approach to troubleshooting is necessary:

• Analyze epitope accessibility issues:

  • Different fixation methods may alter epitope exposure

  • Certain detergents may differentially extract STUB1 from subcellular compartments

  • Try alternative antigen retrieval methods for IHC/IF applications

• Validate antibody specificity in each method:

  • Use STUB1 knockout samples as negative controls for each technique

  • Compare multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

• Consider protein complexes and interactions:

  • STUB1 interactions with chaperones may mask epitopes in native conditions

  • Denaturating conditions in Western blot may reveal epitopes hidden in IF

  • Try crosslinking approaches to stabilize protein complexes before analysis

• Analyze subcellular localization patterns:

  • STUB1 distribution between cytoplasm and nucleus may vary with cellular conditions

  • Use subcellular fractionation to compare with imaging results

  • Include co-localization studies with known STUB1 interactors (HSP70, IFNγ-R1, JAK1)

• Method-specific quantification limitations:

  • Western blot may better reflect total protein levels

  • IF provides spatial information but may be subject to threshold artifacts

  • Consider complementary approaches like flow cytometry for quantitative single-cell analysis

By implementing these strategies, researchers can better understand and reconcile differences observed across experimental platforms.

How can STUB1 antibodies be utilized to investigate IFNγ signaling in cancer immunotherapy research?

STUB1 has emerged as a critical regulator of IFNγ signaling with significant implications for cancer immunotherapy. Researchers can leverage STUB1 antibodies to investigate several key aspects of this relationship:

• Monitoring STUB1-mediated regulation of IFNγ receptor complex:

  • Use co-immunoprecipitation with STUB1 antibodies to isolate and analyze IFNγ-R1/JAK1 complexes

  • Employ proximity ligation assays to visualize STUB1 interactions with receptor components in situ

  • Analyze proteasomal degradation of IFNγ-R1 and JAK1 in response to IFNγ stimulation

• Investigating STUB1 expression in tumor microenvironments:

  • Perform multiplex IHC to correlate STUB1 levels with immune cell infiltration

  • Compare STUB1 expression between responders and non-responders to immune checkpoint blockade

  • Analyze the anticorrelation between STUB1 expression and IFNγ response signatures in patient samples

• Studying STUB1 regulation of tumor cell sensitivity to cytotoxic T cells:

  • Use STUB1 antibodies to validate knockout/knockdown models in tumor cells

  • Correlate STUB1 levels with tumor cell susceptibility to T cell-mediated killing

  • Monitor changes in IFNγ-induced MHC expression and antigen presentation in relation to STUB1 status

• Exploring context-dependent effects in heterogeneous tumors:

  • Analyze STUB1 expression in different regions of tumor samples

  • Compare outcomes between complete STUB1 knockout versus heterogeneous STUB1 expression models

  • Investigate how STUB1 levels influence response to anti-PD-1 therapy in heterogeneous tumor populations

These approaches enable detailed investigation of how STUB1-mediated regulation of IFNγ signaling impacts cancer immunotherapy outcomes, potentially identifying new biomarkers or therapeutic targets.

What are the most effective strategies for studying ubiquitination targets of STUB1 using biotin-conjugated antibodies?

Studying STUB1-mediated ubiquitination requires specialized approaches to capture and analyze these often transient protein modifications. Biotin-conjugated STUB1 antibodies offer particular advantages in these experimental contexts:

• Sequential immunoprecipitation strategy:

  • First IP: Use biotin-conjugated STUB1 antibodies with streptavidin beads to pull down STUB1 complexes

  • Elution under native conditions

  • Second IP: Use antibodies against suspected target proteins (e.g., IFNγ-R1, JAK1)

  • Analyze ubiquitination patterns by Western blot using anti-ubiquitin antibodies

• Site-specific ubiquitination analysis:

  • Focus on key ubiquitination sites like IFNγ-R1 K285 and JAK1 K249

  • Generate lysine-to-arginine mutants of these residues

  • Use biotin-conjugated STUB1 antibodies to compare ubiquitination patterns between wildtype and mutant proteins

• Proteasome inhibition time course analysis:

  • Treat cells with MG132 for different durations

  • Use biotin-conjugated STUB1 antibodies for pulldown

  • Analyze accumulation of ubiquitinated target proteins over time

  • Compare patterns between wildtype and STUB1-deficient cells

• Domain-specific function analysis:

  • Generate cells expressing STUB1 variants (TPR-deficient, UBOX-deficient, E3 ligase dead H260Q)

  • Use biotin-conjugated antibodies against wildtype STUB1 as controls

  • Compare ubiquitination patterns of target proteins across these variants

• Mass spectrometry-based identification of novel targets:

  • Use biotin-conjugated STUB1 antibodies for large-scale immunoprecipitation

  • Analyze samples by mass spectrometry to identify ubiquitinated proteins

  • Validate candidates using targeted approaches in STUB1 wildtype versus knockout cells

These methodological approaches enable comprehensive analysis of STUB1-mediated ubiquitination and the functional consequences for target protein stability and signaling.

How can researchers apply STUB1 antibodies to investigate protein quality control in neurodegenerative diseases?

STUB1's role in protein quality control makes it particularly relevant to neurodegenerative diseases characterized by protein misfolding and aggregation. Researchers can apply STUB1 antibodies to investigate these connections using several methodological approaches:

• Analysis of STUB1 levels and localization in disease models:

  • Compare STUB1 expression between normal and diseased tissue samples

  • Examine co-localization with protein aggregates using multi-label immunofluorescence

  • Assess changes in STUB1 subcellular distribution during disease progression

• Investigation of STUB1 interaction with disease-associated proteins:

  • Perform co-immunoprecipitation using biotin-conjugated STUB1 antibodies

  • Analyze interaction with proteins like tau, α-synuclein, or huntingtin

  • Examine how disease-causing mutations affect these interactions

• Assessment of STUB1 ubiquitination activity toward disease-relevant substrates:

  • Reconstitute ubiquitination reactions in vitro using purified components

  • Compare ubiquitination patterns between wildtype and mutant substrate proteins

  • Use domain-specific STUB1 mutants to dissect molecular requirements

• Analysis of chaperone dependencies in STUB1 function:

  • Examine STUB1-HSP70 interactions using proximity ligation assays

  • Test the effects of HSP70 modulators on STUB1-mediated degradation of aggregation-prone proteins

  • Investigate how cellular stress affects STUB1 chaperone interactions and function

• Therapeutic strategy evaluation:

  • Test compounds that modulate STUB1 activity in cellular and animal models

  • Monitor changes in protein aggregation and clearance using STUB1 antibodies

  • Assess effects on neuronal survival and function in relation to STUB1 activity

These approaches can provide insights into how STUB1 dysfunction may contribute to neurodegenerative pathology and identify potential therapeutic strategies targeting protein quality control mechanisms.

How can STUB1 antibodies contribute to understanding the relationship between inflammation and cancer progression?

Recent findings about STUB1's role in regulating IFNγ signaling suggest broader implications for inflammation-cancer interactions. Researchers can use STUB1 antibodies to explore these connections through several methodological approaches:

• Analysis of STUB1 expression patterns in inflammatory microenvironments:

  • Compare STUB1 levels between inflamed and non-inflamed tissues

  • Correlate STUB1 expression with inflammatory cytokine profiles

  • Examine changes in STUB1 localization during inflammatory responses

• Investigation of STUB1 regulation of inflammatory signaling beyond IFNγ:

  • Use biotin-conjugated STUB1 antibodies to identify novel interaction partners in inflammatory contexts

  • Analyze effects of STUB1 modulation on NF-κB, JAK-STAT, and MAPK pathways

  • Examine how inflammatory stimuli affect STUB1's E3 ligase activity and substrate specificity

• Assessment of STUB1's role in immune cell function:

  • Compare STUB1 expression and function across different immune cell populations

  • Analyze how STUB1 levels affect immune cell activation, cytokine production, and effector functions

  • Investigate STUB1-dependent regulation of immune cell metabolism and survival

• Examination of STUB1 in inflammation-driven cancer models:

  • Monitor STUB1 expression during inflammation-to-cancer transition

  • Analyze how STUB1 status affects tumor initiation in inflammatory contexts

  • Investigate how STUB1-mediated regulation of IFNγ signaling influences anti-tumor immunity

These approaches can illuminate how STUB1 functions at the intersection of inflammation and cancer, potentially identifying new therapeutic opportunities targeting this regulatory axis.

What are the current technical challenges in developing specific assays for STUB1 activity?

Developing reliable assays to measure STUB1's E3 ubiquitin ligase activity presents several technical challenges that researchers must address:

• Distinguishing STUB1 auto-ubiquitination from substrate ubiquitination:

  • STUB1 undergoes self-ubiquitination that can confound substrate activity measurements

  • Recommendation: Use STUB1 catalytic mutants (H260Q) as controls

  • Develop substrate-specific ubiquitination detection methods (e.g., using antibodies recognizing specific ubiquitinated lysines)

• Accounting for chaperone dependencies:

  • STUB1 activity relies on interactions with chaperones like HSP70

  • Recommendation: Include defined chaperone components in in vitro assays

  • Monitor chaperone levels and activity states in cellular assays

• Capturing the transient nature of ubiquitination events:

  • Ubiquitinated substrates are rapidly degraded by the proteasome

  • Recommendation: Use proteasome inhibitors with careful time course analysis

  • Develop reporters based on stabilized substrate variants

• Addressing context-dependent substrate specificity:

  • STUB1 targets different substrates depending on cellular context

  • Recommendation: Compare activity across relevant cell types and conditions

  • Develop cell-type specific activity assays

• Standardizing activity measurements:

  • Different assay formats yield variable results

  • Recommendation: Establish reference standards and positive controls

  • Develop quantitative readouts with appropriate normalization

By addressing these challenges, researchers can develop more robust and informative assays for STUB1 activity, enabling better understanding of its roles in health and disease.