BCAP31 Antibody

What is BCAP31 and why is it an important research target?

BCAP31 (B-Cell Receptor-Associated Protein 31) is a 28-31 kDa membrane protein predominantly located in the endoplasmic reticulum (ER). It plays crucial roles in protein trafficking, calcium homeostasis, and apoptosis regulation. As a chaperone protein, it assists in correctly folding and transporting newly synthesized proteins within the cell . Recent research has identified BCAP31 as a potential prognostic marker in non-small-cell lung cancer (NSCLC), highlighting its relation to cancer metastasis through the Akt/m-TOR/p70S6K pathway . Additionally, mutations in the BCAP31 gene have been linked to X-linked chondrodysplasia punctata type 2 (CDPX2), a disorder characterized by skeletal abnormalities and developmental defects .

Which applications are most suitable for BCAP31 antibody detection?

BCAP31 antibodies have demonstrated efficacy across multiple applications:

The optimal application choice depends on your research question, with WB being most commonly used for expression level analysis and IF/IHC for localization studies .

How can I optimize the specificity of BCAP31 antibody detection in Western blotting?

For optimal specificity in Western blotting:

• Antibody selection: Choose antibodies validated for WB with reactivity to your species of interest. Monoclonal antibodies (like clone 1502CT208-31-16) offer high specificity .

• Sample preparation: Use appropriate lysis buffers with protease inhibitors. BCAP31 is membrane-associated, so ensure complete solubilization.

• Dilution optimization: Start with a 1:5000 dilution and adjust based on signal-to-noise ratio .

• Controls: Include a positive control (A431, HeLa, or HEK-293 cells have been validated) and consider a BCAP31 knockdown/knockout negative control .

• Detection method: PVDF membranes with HRP-conjugated secondary antibodies have been successfully used for BCAP31 detection at approximately 28-31 kDa .

• Reducing conditions: BCAP31 detection is typically performed under reducing conditions .

How does epitope specificity influence BCAP31 antibody selection for different experimental aims?

Epitope specificity is critical when selecting BCAP31 antibodies, as different epitopes yield different insights:

When studying BCAP31's membrane topology, antibodies specific to the C-terminal domain (residues 208-217) are particularly valuable, as research has shown this domain is exposed on the cell surface, contrary to earlier predictions . For investigations of BCAP31 cleavage during apoptosis, antibodies recognizing regions around the caspase recognition sites (Asp164-Gly165 and Asp238-Gly239) are most informative .

What strategies can resolve discrepancies in BCAP31 subcellular localization across different studies?

Discrepancies in BCAP31 localization findings can be addressed through:

• Complementary techniques: Combine immunofluorescence with subcellular fractionation and Western blotting to confirm localization patterns.

• Antibody validation: Use multiple antibodies targeting different epitopes. Some studies used monoclonal antibodies 297-D4 and 144-A8 to confirm novel membrane topology with C-terminal domain exposure on cell surface .

• Cell type considerations: BCAP31 localization varies by cell type. Examine both endogenous expression and tagged constructs in relevant cell lines.

• Co-localization studies: Use established markers for ER (calnexin), Golgi (GM130), plasma membrane (Na+/K+ ATPase), and other compartments alongside BCAP31 staining .

• Super-resolution microscopy: For precise localization, techniques like STORM or STED microscopy can resolve ER-plasma membrane contact sites where BCAP31 may be enriched.

• Live-cell imaging: For dynamic studies of BCAP31 trafficking between compartments, especially during ER stress responses.

The unexpected finding that BCAP31's C-terminal domain can be exposed on the cell surface of embryonic stem cells suggests previously unknown functions and demonstrates why multiple methodological approaches are essential .

How can researchers distinguish between different BCAP31 cleavage products during apoptosis studies?

Distinguishing BCAP31 cleavage products requires precise methodological approaches:

• Antibody selection: Use antibodies that recognize different domains:

  • N-terminal specific antibodies for p20 N-terminal fragment

  • C-terminal specific antibodies for p27 fragment

  • Full-length specific antibodies to monitor intact protein depletion

• Time-course experiments: Monitor cleavage events temporally after apoptosis induction, as early phase typically shows initial cleavage at Asp238 site, while later phases show additional cleavage at Asp164 .

• Caspase inhibitors: Use specific inhibitors (caspase-8 vs. caspase-1) to differentiate between cleavage pathways.

• Site-directed mutagenesis: Create BCAP31 constructs with mutated cleavage sites (D164A, D238A, or double mutants) to confirm fragment identities.

• Size differentiation: Use high-percentage (12-15%) SDS-PAGE gels to clearly separate p20 (~20 kDa) from p27 (~27 kDa) fragments.

• Subcellular fractionation: Cleaved forms may redistribute within cells; membrane and cytosolic fractions should be analyzed separately.

What are the cross-species reactivity patterns of commonly available BCAP31 antibodies?

BCAP31 antibodies show diverse species reactivity patterns:

How can researchers validate BCAP31 antibody specificity in their model systems?

Thorough validation strategies include:

• Positive controls: Use cell lines with known high BCAP31 expression (A431, HeLa, HEK-293, MCF-7) .

• Genetic approaches:

  • siRNA or shRNA knockdown of BCAP31

  • CRISPR/Cas9 knockout of BCAP31

  • Overexpression of tagged BCAP31 constructs

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm signal suppression.

• Multiple antibodies: Compare staining patterns using antibodies targeting different epitopes.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm pulled-down proteins.

• Cross-reactivity testing: Test reactivity against related family members (especially BAP29) in overexpression systems.

• Specificity in multiple applications: Confirm consistent molecular weight in WB, expected subcellular localization in IF, and appropriate tissue expression patterns in IHC .

How can BCAP31 antibodies be used to investigate its role in cancer progression?

BCAP31 has emerging significance in cancer research, particularly in NSCLC:

Based on findings that BCAP31 functions as a cancer/testis antigen-like protein, antibodies can help elucidate its potential as both a biomarker and therapeutic target .

What methodological approaches are optimal for studying BCAP31's role in protein quality control and trafficking?

To investigate BCAP31's role in protein quality control:

• Co-immunoprecipitation: Use BCAP31 antibodies (0.5-4.0 μg per 1-3 mg lysate) to pull down complexes and identify interacting client proteins .

• Pulse-chase experiments: Track client protein maturation and trafficking after BCAP31 manipulation, using antibodies against both BCAP31 and client proteins.

• BCAP31-client protein co-localization: Perform double immunofluorescence with BCAP31 antibodies (1:50-1:500 dilution) and antibodies against client proteins like CFTR, class I MHC, or cellubrevin .

• ER retention assays: Monitor client protein localization after BCAP31 depletion to determine if BCAP31 promotes forward transport or ER retention.

• Glycosylation status analysis: Use BCAP31 antibodies in conjunction with endoglycosidase treatments to assess client protein maturation.

• Live cell imaging: Track GFP-tagged client proteins with BCAP31 manipulation to observe dynamic trafficking events.

• ER stress induction: Monitor BCAP31-client interactions during chemical induction of ER stress (tunicamycin, thapsigargin) using proximity ligation assays with specific antibodies.

How can researchers investigate the novel membrane topology of BCAP31 on the cell surface?

To explore BCAP31's unexpected cell surface topology:

• Epitope accessibility assays: Use monoclonal antibodies targeting different domains (like 297-D4 and 144-A8 for C-terminal detection) on non-permeabilized cells to map exposed regions .

• Deletion mutant analysis: Create GST-fused BAP31 mutant proteins with serial C-terminal deletions to precisely map epitopes recognized by different antibodies .

• Surface biotinylation: Perform cell surface protein biotinylation followed by BCAP31 immunoprecipitation to confirm surface localization.

• Protease protection assays: Treat intact cells with proteases that cannot penetrate the membrane, then analyze BCAP31 fragments with domain-specific antibodies.

• Cell type comparison: Compare antibody accessibility in pluripotent stem cells versus differentiated cells, as novel topology was first observed in human embryonic stem cells .

• Functional studies: Investigate potential signaling roles of surface-exposed BCAP31 using antibodies as blocking agents or stimulating agents.

• Topology modeling: Integrate experimental findings with computational predictions to create refined models of BCAP31 membrane topology.

The unexpected finding that the C-terminal domain (residues 208-217) is exposed on the cell surface contradicts previous topology models and suggests novel functions for BCAP31 beyond its established ER roles .

How can researchers address common issues with BCAP31 antibodies in Western blotting?

Common Western blot issues with BCAP31 antibodies can be resolved through:

For specific antibody performance, consult validation galleries from manufacturers like Proteintech that show expected banding patterns in various cell lines (A431, HEK-293, HeLa, MCF-7) .

What methodological adaptations are required for successful immunoprecipitation of BCAP31?

For successful BCAP31 immunoprecipitation:

• Lysis conditions: Use mild detergents (0.5-1% NP-40 or Triton X-100) in TBS or PBS with protease inhibitors to maintain membrane protein interactions.

• Antibody selection: Choose antibodies specifically validated for IP applications. Polyclonal antibodies often perform better in IP than monoclonals .

• Antibody amount: Start with 0.5-4.0 μg antibody per 1-3 mg of total protein lysate . Titrate to optimize signal-to-noise ratio.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Incubation conditions: Perform antibody binding overnight at 4°C with gentle rotation to maintain native protein complexes.

• Washing stringency: Balance between removing non-specific binding and preserving specific interactions; typically 3-5 washes with decreasing detergent concentrations.

• Elution optimization: Use either low pH glycine buffers or SDS-sample buffer depending on downstream applications.

• Co-IP considerations: For interaction studies, cross-linking may help stabilize transient interactions before cell lysis.

A431 cells have been validated as positive controls for BCAP31 IP, with approximately 28 kDa band detection on subsequent Western blots .

What strategies can improve immunohistochemical detection of BCAP31 in different tissue types?

For optimal BCAP31 IHC results:

• Antigen retrieval methods: Compare heat-induced epitope retrieval using:

  • TE buffer pH 9.0 (recommended as primary method)

  • Citrate buffer pH 6.0 (alternative method)

• Antibody dilution optimization: Start with 1:50-1:500 dilution range and optimize for each tissue type .

• Detection systems:

  • DAB chromogen for brightfield microscopy

  • Fluorescent secondary antibodies for multi-channel co-localization

• Positive control tissues: Include validated tissues like:

  • Human colon cancer tissue

  • Human breast cancer tissue

  • Human placenta tissue

• Blocking optimization: For tissues with high background, extend blocking with:

  • 5-10% normal serum from secondary antibody host species

  • Addition of 0.1-0.3% Triton X-100 for improved penetration

  • Avidin/biotin blocking for tissues with endogenous biotin

• Signal amplification: For low abundance detection, consider tyramide signal amplification systems.

• Multi-labeling considerations: When co-staining with other markers, select antibodies from different host species or use directly conjugated primary antibodies.

BCAP31 expression patterns vary by tissue type, with particularly strong staining observed in secretory and metabolically active tissues, requiring tissue-specific protocol optimization .

How can BCAP31 antibodies contribute to understanding ER stress and unfolded protein response pathways?

BCAP31 antibodies enable several approaches to UPR research:

• Stress-induced relocalization: Track BCAP31 subcellular redistribution during ER stress using IF with co-staining for UPR sensors (IRE1α, PERK, ATF6).

• Interaction dynamics: Use co-IP with BCAP31 antibodies to identify stress-dependent changes in protein interactions.

• Post-translational modifications: Develop phospho-specific antibodies to monitor BCAP31 phosphorylation status during UPR activation.

• BCAP31 cleavage monitoring: Track caspase-mediated BCAP31 processing during ER stress-induced apoptosis using domain-specific antibodies.

• ChIP-seq applications: For potential transcriptional regulation studies if nuclear BCAP31 fragments are detected.

• Proximity-based proteomics: Combine BCAP31 antibodies with BioID or APEX2 approaches to map stress-dependent interaction networks.

• Patient-derived samples: Compare BCAP31 expression and processing in tissues from patients with ER stress-related diseases using validated antibodies.

These approaches can help elucidate how BCAP31 contributes to calcium homeostasis disruption and apoptotic signaling during prolonged ER stress .

What novel insights might be gained from investigating BCAP31's role in rare genetic disorders?

BCAP31 antibodies enable critical investigations of its role in genetic disorders:

• X-linked chondrodysplasia punctata (CDPX2): Use tissue-specific IHC to examine:

  • BCAP31 expression patterns in affected tissues

  • Accumulation of misfolded client proteins

  • Alterations in ER morphology and stress markers

• Mutation impact assessment:

  • Compare wildtype vs. mutant BCAP31 localization using IF

  • Analyze protein stability and half-life differences

  • Assess chaperone function impairment through client protein interactions

• Developmental studies:

  • Track BCAP31 expression during embryonic development in model organisms

  • Correlate expression with developmental defects in patient-derived samples

• iPSC disease modeling:

  • Generate iPSCs from patient cells

  • Differentiate into relevant cell types (chondrocytes, neurons)

  • Compare BCAP31 function between patient and control cells

• Therapeutic screening:

  • Use BCAP31 antibodies to monitor correction of cellular phenotypes

  • Assess efficacy of chaperone-enhancing compounds

  • Track responses to ER stress modulators

These approaches could reveal mechanistic connections between BCAP31 dysfunction and developmental abnormalities, potentially identifying therapeutic targets for rare disorders associated with BCAP31 mutations .

What emerging technologies might enhance BCAP31 antibody applications in future research?

Emerging technologies offering new opportunities for BCAP31 research include:

• Super-resolution microscopy: Techniques like STORM, PALM, and STED can resolve BCAP31 distribution within ER subdomains and at ER-organelle contact sites with nanometer precision.

• Intrabodies and nanobodies: Developing small antibody fragments against BCAP31 for live-cell imaging and acute protein inhibition.

• Spatial transcriptomics integration: Combining BCAP31 IHC with spatial transcriptomics to correlate protein expression with local transcriptional environments.

• Multiplex imaging: Using multiplexed ion beam imaging (MIBI) or cyclic immunofluorescence (CycIF) to simultaneously visualize BCAP31 with dozens of other proteins.

• Cryo-electron tomography: Visualizing BCAP31's native membrane organization and protein complexes at near-atomic resolution.

• Targeted protein degradation: Developing BCAP31-directed PROTACs or dTAGs for rapid, inducible protein depletion without genetic manipulation.

• Microfluidic applications: High-throughput single-cell analysis of BCAP31 expression and co-expression patterns across populations.

• AI-enhanced image analysis: Deep learning approaches to identify subtle BCAP31 distribution patterns that correlate with cellular states or disease progression.