HSPA1B Antibody, FITC conjugated

What is HSPA1B and what cellular functions does it perform?

HSPA1B (Heat Shock 70kDa Protein 1B) is a 70kDa member of the heat shock protein 70 family. This intronless gene encodes a protein that works cooperatively with other chaperones to stabilize existing proteins against aggregation and mediate the folding of newly translated polypeptides in both the cytosol and organelles . HSPA1B functions by recognizing nonnative conformations of other proteins, binding extended peptide segments with hydrophobic character that are exposed during translation, membrane translocation, or following stress-induced damage . Additionally, HSPA1B plays an essential role in the STUB1-mediated ubiquitination and degradation of FOXP3 in regulatory T-cells (Treg) during inflammation, highlighting its importance in immune regulation .

What applications are supported by FITC-conjugated HSPA1B antibodies?

FITC-conjugated HSPA1B antibodies support multiple research applications across cellular and molecular biology. According to technical specifications, these antibodies can be utilized in:

The FITC conjugation (Excitation = 495 nm, Emission = 519 nm) enables direct visualization in fluorescence-based applications without the need for secondary antibodies, streamlining experimental workflows .

What are the key differences between available HSPA1B antibody formulations?

Several formulations of HSPA1B antibodies exist, varying in host species, clonality, and target epitopes:

Researchers should select the appropriate antibody format based on their specific experimental requirements, target species, and detection methodology .

How should HSPA1B subcellular localization studies be designed?

When designing subcellular localization studies for HSPA1B, researchers should consider its documented localization patterns in both the cytoplasm and nucleus . For optimal results:

• Use paraformaldehyde fixation (4%) followed by appropriate permeabilization with 0.1-0.5% Triton X-100 to preserve cellular architecture.

• Counter-stain with organelle-specific markers (e.g., DAPI for nuclei, MitoTracker for mitochondria) to establish precise localization patterns.

• Consider that HSPA1B localization may shift under stress conditions, necessitating appropriate experimental controls (heat shock, oxidative stress, etc.).

• Account for the FITC excitation/emission spectrum (495nm/519nm) when designing multi-color imaging experiments to avoid spectral overlap .

• Implement z-stack imaging to differentiate between true co-localization and superimposition of signals from different focal planes.

The choice between monoclonal and polyclonal antibodies will affect specificity and signal strength, with monoclonals like mouse IgG2b kappa clone HSPA1B/7623 offering high specificity for particular epitopes .

What protocols optimize FITC-conjugated HSPA1B antibody performance in flow cytometry?

For flow cytometry applications using FITC-conjugated HSPA1B antibodies:

• Cell Preparation:

  • For intracellular staining, use a fixation/permeabilization buffer compatible with the preservation of HSPA1B epitopes.

  • Maintain cell concentration at 1×10^6 cells/100μL to ensure optimal antibody binding.

• Antibody Titration:

  • Perform titration experiments to determine optimal concentration (typically starting at manufacturer's recommendation and testing 2-fold dilutions).

  • Evaluate staining index (signal-to-noise ratio) for each concentration.

• Compensation Controls:

  • Include single-stained controls for each fluorophore in multi-parameter panels.

  • Account for FITC spectral overlap with PE and other fluorophores in the 530nm emission range.

• Analysis Considerations:

  • Include FMO (Fluorescence Minus One) controls to determine accurate gating boundaries.

  • Consider stress-induced upregulation of HSPA1B when interpreting results from experimental vs. control samples.

• Storage Precautions:

  • Store antibody at -20°C or -80°C for long-term storage to prevent degradation .

  • Avoid repeated freeze-thaw cycles as these may reduce antibody functionality .

How can researchers investigate HSPA1B involvement in STUB1-mediated ubiquitination pathways?

To investigate HSPA1B's role in STUB1-mediated ubiquitination and degradation of FOXP3 in regulatory T-cells:

• Co-Immunoprecipitation Strategy:

  • Use FITC-conjugated HSPA1B antibodies for pull-down experiments followed by Western blot analysis for STUB1 and FOXP3.

  • Confirm interactions under different inflammatory conditions to assess context-dependent associations.

• Ubiquitination Assays:

  • Employ in vitro ubiquitination assays with purified components including HSPA1B, STUB1, and FOXP3.

  • Use immunoblotting with anti-ubiquitin antibodies to assess ubiquitination levels.

• Functional Validation:

  • Implement HSPA1B knockdown/knockout approaches to assess effects on FOXP3 stability.

  • Compare FOXP3 protein levels and Treg function in the presence/absence of HSPA1B under inflammatory stimuli.

• Fluorescence Microscopy:

  • Utilize FITC-conjugated HSPA1B antibodies to visualize co-localization with STUB1 and FOXP3 in Tregs.

  • Quantify co-localization coefficients under different inflammatory conditions.

• Protein Degradation Kinetics:

  • Employ cycloheximide chase assays with and without HSPA1B to assess FOXP3 degradation rates.

  • Quantify results using appropriate image analysis software for fluorescence intensity measurements.

These approaches leverage the FITC conjugation for direct visualization while enabling mechanistic investigation of this critical immune regulatory pathway .

What are the recommended protocols for immunohistochemistry using FITC-conjugated HSPA1B antibodies?

For optimal immunohistochemistry results with FITC-conjugated HSPA1B antibodies:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours.

  • Process and embed in paraffin following standard protocols.

  • Cut sections at 4-6μm thickness onto positively charged slides.

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

  • Heat at 95-100°C for 20 minutes followed by cooling to room temperature.

• Blocking and Antibody Application:

  • Block non-specific binding with 5-10% normal serum from the same species as the secondary antibody.

  • Apply FITC-conjugated HSPA1B antibody at experimentally determined optimal dilution (typically starting at 1:100-1:500).

  • Incubate in a humidified chamber at 4°C overnight or at room temperature for 1-2 hours.

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1μg/mL) for 5-10 minutes.

  • Mount with anti-fade mounting medium to preserve FITC fluorescence.

• Controls:

  • Include positive controls (tissues known to express HSPA1B).

  • Include negative controls (primary antibody omitted or non-specific antibody of same isotype).

Optimal dilutions should be experimentally determined for each specific application and tissue type .

How should Western blotting protocols be modified for FITC-conjugated HSPA1B antibodies?

When performing Western blotting with FITC-conjugated HSPA1B antibodies:

• Sample Preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors.

  • Heat samples at 70°C (instead of 95°C) for 10 minutes to preserve HSPA1B epitopes.

  • Load 20-50μg of total protein per lane.

• Gel Electrophoresis and Transfer:

  • Separate proteins on 10% SDS-PAGE gels.

  • Transfer to PVDF membranes (preferred over nitrocellulose due to better protein retention).

• Blocking and Antibody Incubation:

  • Block membranes with 5% non-fat dry milk or 3-5% BSA in TBST.

  • Dilute FITC-conjugated HSPA1B antibody in blocking buffer at 1:500-1:2000 .

  • Incubate overnight at 4°C with gentle agitation.

• Detection Strategies:

  • Direct Fluorescence: Visualize directly using a fluorescence imager with appropriate filters (Ex: 495nm, Em: 519nm).

  • Enhanced Detection: For higher sensitivity, consider anti-FITC HRP-conjugated antibody followed by chemiluminescence detection.

• Important Considerations:

  • Protect membranes from light during all steps to prevent photobleaching of FITC.

  • Include positive controls (cells expressing high levels of HSPA1B).

  • Expected band size for HSPA1B is approximately 70kDa.

This protocol should yield specific detection of HSPA1B in various sample types, with recommended dilutions optimized specifically for Western blot applications .

What storage and handling precautions ensure maximum stability of FITC-conjugated HSPA1B antibodies?

To maintain optimal functionality of FITC-conjugated HSPA1B antibodies:

Upon delivery, it is recommended to immediately aliquot the antibody into single-use volumes (typically 10-20μL) and store at -80°C for maximum long-term stability . When handling, minimize exposure to light as FITC is susceptible to photobleaching, which can reduce signal intensity in fluorescence-based applications.

How can researchers address non-specific binding and background issues with FITC-conjugated HSPA1B antibodies?

When encountering high background or non-specific binding:

• Optimize Blocking Conditions:

  • Increase blocking agent concentration (5-10% normal serum or BSA).

  • Extend blocking time to 1-2 hours at room temperature.

  • Consider adding 0.1-0.3% Triton X-100 to blocking buffer for intracellular applications.

• Antibody Dilution and Incubation:

  • Perform titration experiments to determine optimal antibody concentration.

  • Consider reducing primary antibody concentration if background is high.

  • Extend washing steps (5-6 washes of 5 minutes each) with TBST or PBS-T.

• Sample-Specific Considerations:

  • For tissues with high autofluorescence, treat with 0.1-1% sodium borohydride for 5-10 minutes prior to antibody incubation.

  • Include quenching step with 0.3% H₂O₂ in methanol to reduce endogenous peroxidase activity for IHC applications.

• FITC-Specific Strategies:

  • Use a mounting medium with anti-fade properties to reduce photobleaching.

  • Adjust microscope settings to optimize signal-to-noise ratio (reduce gain if background is high).

  • Consider spectral unmixing algorithms if autofluorescence occurs in the FITC emission range.

• Validation Controls:

  • Include isotype control antibodies (same host species and isotype, FITC-conjugated) to identify non-specific binding.

  • Perform pre-absorption controls with recombinant HSPA1B protein to confirm specificity.

These approaches systematically address the most common sources of background and non-specificity when working with FITC-conjugated antibodies .

What strategies can validate the specificity of HSPA1B antibody detection in experimental systems?

To ensure specificity of HSPA1B antibody detection:

• Epitope Confirmation:

  • Compare results using antibodies targeting different HSPA1B epitopes (e.g., AA 400-600 region vs. AA 531-611 ).

  • Verify recognition of the specific isoform vs. other heat shock protein family members.

• Genetic Validation:

  • Perform parallel experiments in HSPA1B knockout/knockdown systems.

  • Observe signal reduction or elimination in depleted samples compared to controls.

• Recombinant Protein Controls:

  • Use purified recombinant HSPA1B as a positive control in Western blots.

  • Perform competition assays by pre-incubating antibody with excess recombinant protein.

• Cross-Species Validation:

  • Test antibody performance across specified reactive species (human, mouse, rat, cow) as indicated in product documentation .

  • Confirm alignment of targeted epitope sequences across species.

• Multi-Method Confirmation:

  • Verify HSPA1B detection using complementary techniques (e.g., Western blot and immunofluorescence).

  • Compare results between different antibody clones (e.g., HSPA1B/7623 vs. other available clones).

These validation approaches provide multiple lines of evidence for antibody specificity, which is essential for confident interpretation of experimental results involving HSPA1B detection .

What are the key considerations for quantitative analysis of HSPA1B expression using FITC-conjugated antibodies?

For accurate quantitative analysis of HSPA1B expression:

• Signal Calibration:

  • Use quantitative fluorescence standards with known fluorophore concentrations.

  • Establish standard curves relating fluorescence intensity to protein quantity.

• Image Acquisition Parameters:

  • Maintain consistent exposure settings across all samples and controls.

  • Avoid pixel saturation by using exposure times that keep signal within the linear range.

  • Collect images at optimal bit depth (minimum 12-bit) to capture subtle differences in expression.

• Background Correction:

  • Implement appropriate background subtraction methods.

  • Use rolling ball algorithm for uneven background in immunofluorescence.

  • Measure adjacent regions for local background in tissue sections.

• Technical Considerations:

  • Account for FITC photobleaching by minimizing exposure time during imaging.

  • Image all samples within the same session to minimize day-to-day variations.

  • Include internal reference standards in each experiment.

• Analytical Approaches:

  • Use specialized software for quantification (ImageJ/FIJI, CellProfiler, etc.).

  • Apply appropriate statistical tests based on data distribution.

  • Present results as relative expression normalized to housekeeping proteins or total protein content.

These considerations ensure reliable quantitative assessment of HSPA1B expression levels across experimental conditions and biological samples .

How can FITC-conjugated HSPA1B antibodies be utilized in exosome characterization studies?

FITC-conjugated HSPA1B antibodies offer valuable tools for exosome research:

• Exosome Marker Identification:

  • HSPA1B (Hsp70) serves as an established exosome-specific marker alongside CD63 and TSG101 .

  • Use FITC-conjugated HSPA1B antibodies in flow cytometry or immunoblotting to confirm exosomal identity.

• Multi-Parameter Exosome Characterization:

  • Combine FITC-conjugated HSPA1B antibodies with antibodies against other exosome markers labeled with compatible fluorophores.

  • Perform co-localization analysis to confirm presence of multiple markers on the same exosomal populations.

• Functional Studies:

  • Track exosome uptake and distribution using FITC-labeled antibodies against HSPA1B on the exosome surface.

  • Monitor changes in exosomal HSPA1B content under different physiological or stress conditions.

• Technical Workflow:

  • Isolate exosomes using ultracentrifugation, size exclusion chromatography, or commercial kits.

  • Validate exosome preparation by NTA (Nanoparticle Tracking Analysis) or DLS (Dynamic Light Scattering).

  • Perform Western blotting using HSPA1B antibodies at 1:500-1:2000 dilution to confirm marker presence .

• Recent Research Applications:

  • Studies have successfully used this approach to characterize exosomes in OGD/R (Oxygen-Glucose Deprivation/Reperfusion) and BBR (Berberine) models .

  • HSPA1B detection has become a standard validation step in exosome characterization workflows.

This emerging application leverages the specificity of HSPA1B antibodies for standardized exosome characterization protocols .

What role do HSPA1B antibodies play in investigating stress response mechanisms?

FITC-conjugated HSPA1B antibodies enable detailed investigation of stress response mechanisms:

• Stress-Induced Translocation:

  • Monitor HSPA1B translocation between cytoplasm and nucleus during stress responses .

  • Track temporal dynamics using time-lapse fluorescence microscopy with direct FITC visualization.

• Protein-Protein Interaction Networks:

  • Use FITC-conjugated HSPA1B antibodies in proximity ligation assays to identify stress-induced interaction partners.

  • Quantify changes in interaction profiles under various stress conditions (heat, oxidative, ER stress).

• Client Protein Identification:

  • Employ HSPA1B antibodies in co-immunoprecipitation followed by mass spectrometry to identify stress-dependent client proteins.

  • Validate interactions using fluorescence co-localization of FITC-labeled HSPA1B with candidate proteins.

• Stress Granule Association:

  • Investigate HSPA1B recruitment to stress granules under stress conditions.

  • Perform co-localization studies with stress granule markers (G3BP1, TIA-1) to quantify recruitment kinetics.

• Therapeutic Targeting:

  • Screen compounds that modulate HSPA1B function or localization using high-content imaging approaches.

  • Quantify changes in HSPA1B-dependent stress responses as potential therapeutic interventions.

These applications provide mechanistic insights into how HSPA1B coordinates cellular responses to stress, offering potential therapeutic targets for stress-related pathologies .