HSPB8 Antibody

What is HSPB8 and why is it important in research?

HSPB8 (Heat shock protein B8, also known as HSP22) is a 22 kDa member of the small heat shock protein family that functions as a molecular chaperone. It contains one alpha-crystalline domain (aa 93-170) that mediates protein-protein interactions and forms homo-oligomers and heterodimers with other heat shock proteins like HSP27 and HSPB7 . HSPB8 is particularly important in research because:

• It participates in proteolytic processes including proteasome and autophagy-mediated degradation of misfolded proteins

• It forms complexes with BAG3 (Bcl-2-associated athanogene 3) that regulate macroautophagy

• It shows altered expression in various pathological conditions including cancer

• It plays significant roles in neurodegenerative diseases through its chaperone functions

Research on HSPB8 is advancing our understanding of protein quality control systems, stress responses, and pathological mechanisms in multiple diseases.

What applications are HSPB8 antibodies commonly used for?

Based on published research and manufacturer specifications, HSPB8 antibodies are validated for multiple applications:

For optimal results, antibody concentration should be titrated for each specific application and sample type. Published research indicates that HSPB8 antibodies have been successfully used to detect the protein in multiple cell lines including Neuro-2a, SGC-7901, HEK-293, and in tissues including brain, heart, kidney, lung, and various cancer tissues .

How should I design experiments to study HSPB8 expression changes in disease models?

When designing experiments to evaluate HSPB8 expression changes in disease models, consider the following methodological approach:

• Model selection: Based on published research, both cell culture and animal models are suitable. For cancer studies, established cell lines like T24 (bladder cancer) or MCF-7 (breast cancer) have shown reliable HSPB8 expression. For in vivo models, mouse models have been extensively validated .

• Controls: Include both positive and negative controls. For example, tissues known to express HSPB8 highly (heart and brain tissue) can serve as positive controls .

• Multiple detection methods: Combine complementary techniques:

  • qRT-PCR for mRNA expression

  • Western blot for protein level quantification

  • Immunohistochemistry for spatial localization

  • Co-immunoprecipitation for interaction studies

• Time course design: HSPB8 expression can change dynamically over time. In the optic nerve crush model, researchers observed variable HSPB8 expression at different timepoints post-injury .

• Statistical analysis: Use appropriate statistical methods for your experimental design. Most HSPB8 studies employed ANOVA with post-hoc tests for multiple comparisons and t-tests for binary comparisons .

For disease-specific relevance, studies have successfully used these approaches to examine HSPB8 in bladder cancer , breast cancer , neurodegenerative conditions , and other pathologies.

What are the key considerations for HSPB8 knockdown or overexpression experiments?

When manipulating HSPB8 expression in experimental systems, several methodological considerations are crucial:

For HSPB8 knockdown:

• Silencing efficiency validation: In published studies, researchers evaluated HSPB8 silencing efficiency at both mRNA and protein levels. Using RT-PCR, significant reduction of HSPB8 mRNA was detectable 1 day post-transfection, with complete silencing achieved by day 3 and lasting up to 5 days .

• siRNA design: Published studies have successfully used specific siRNA targeting HSPB8. The reported silencing efficiency was validated by both RT-PCR and Western blot .

• Alternative approaches: For in vivo models, researchers have used AAV2-shHSPB8 viral vectors for stable knockdown. In a mouse optic nerve injury model, intravitreal injection of AAV2-shHSPB8 effectively reduced HSPB8 expression .

For HSPB8 overexpression:

• Expression vector selection: Studies have used pCI-NEO-HSPB8 plasmid for transient transfection in cell lines like MCF-7 .

• Functional validation: Beyond confirming expression levels, researchers have evaluated phenotypic changes such as cell cycle distribution. In MCF-7 cells, HSPB8 overexpression correlated with minor changes in cell cycle distribution, including reduction in G0/G1 phase cells (63.64% vs. 68.33% in control) and slight increase in G2/M phase cells (19.17% vs. 17.06%) .

• Expression timing: Peak expression typically occurs 48-72 hours post-transfection, which is important for experimental timeline planning.

In both approaches, proper controls (empty vectors, scrambled siRNA) are essential, as demonstrated in the referenced studies .

What are the optimal protocols for Western blot detection of HSPB8?

Based on published research methodologies, the following protocol optimizations have proven successful for Western blot detection of HSPB8:

Sample preparation:

• For cell lysates: Standard RIPA buffer works well for most applications, but some researchers found that gel loading buffer (GLB: 120 mM Tris-HCl pH 6.8; 2% SDS; 2 mM DTT; 20% glycerol; 5% 2-mercaptoethanol; 0.01% bromophenol blue) provided more effective isolation of HSPB8 from zebrafish embryos .

• For tissue homogenates: Homogenize tissues in extraction buffer and centrifuge at high speed (>12,000 × g) to obtain clear supernatant .

Electrophoresis and transfer:

• Use 10-12% SDS-PAGE gels for optimal separation of HSPB8 (20-22 kDa) .

• Transfer to 0.2 μm nitrocellulose or 0.42 μm PVDF membranes at standard conditions .

Immunodetection:

• Recommended dilutions: 1:500-1:3000 for most HSPB8 antibodies .

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Overnight at 4°C for optimal signal-to-noise ratio.

• Secondary antibody: Anti-rabbit or anti-mouse HRP-conjugated antibodies depending on the primary antibody host species.

Visualization:

• Enhanced chemiluminescence detection systems like ChemiDoc XRS+ (Bio-Rad) or G-Box (Syngene) have been successfully used .

Special considerations:

• HSPB8 has an observed molecular weight of 20-22 kDa , which can vary slightly depending on post-translational modifications.

• Positive controls: Heart and brain tissues have high endogenous expression and serve as good positive controls .

How can I optimize immunohistochemistry (IHC) protocols for HSPB8 detection in different tissue types?

Optimizing IHC for HSPB8 detection requires tissue-specific considerations:

Antigen retrieval:

• For human tissues: TE buffer pH 9.0 is the primary recommended method for HSPB8 detection .

• Alternative method: Citrate buffer pH 6.0 has also been used successfully when TE buffer doesn't yield optimal results .

Antibody dilution and incubation:

• Recommended dilution range: 1:20-1:200 for most IHC applications .

• Incubation time: Overnight at 4°C yields the best signal-to-noise ratio.

Tissue-specific validations:
HSPB8 antibodies have been successfully used for IHC in multiple human tissues including:

• Heart tissue

• Brain tissue

• Kidney tissue

• Lung tissue

• Ovary tissue

• Placenta tissue

• Skin tissue

• Spleen tissue

• Testis tissue

Detection systems:

• DAB (3,3'-diaminobenzidine) chromogenic detection with hematoxylin counterstaining has been widely used.

• For co-localization studies, fluorescent secondary antibodies can be employed.

Controls:

• Positive control: Include tissues known to express HSPB8 (heart, brain).

• Negative control: Primary antibody omission or isotype control.

• Validation: When possible, verify IHC results with complementary methods like Western blot or RT-PCR.

What are the methodological considerations for co-immunoprecipitation using HSPB8 antibodies?

Co-immunoprecipitation (Co-IP) with HSPB8 antibodies requires specific methodological attention to detect protein-protein interactions:

Sample preparation:

• Cell or tissue lysis: Use a gentle, non-denaturing lysis buffer that preserves protein-protein interactions. Published protocols have used IP lysis buffer containing protease inhibitors .

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

• Protein quantification: Adjust protein concentration to ensure comparable amounts between samples.

Immunoprecipitation:

• Antibody immobilization: Some researchers have successfully used amine-reactive resin for covalent coupling of anti-HSPB8 antibodies .

• Controls: Include a negative control column with resin only and a positive control with a known interacting partner (like anti-BAG3 antibody) .

• Incubation: Overnight incubation at 4°C maximizes binding efficiency.

• Washing: Multiple gentle washes with buffer to remove non-specifically bound proteins.

• Elution: Careful elution with appropriate buffer to isolate protein complexes.

Detection and analysis:

• Western blot analysis of the eluates using antibodies against HSPB8 and suspected interaction partners.

• For unbiased discovery of novel interactors, liquid chromatography-mass spectrometry (LC-MS) of the eluates has been employed successfully .

Known interactions to validate results:

• HSPB8 forms complexes with BAG3, which can serve as a positive control .

• HSPB8 also interacts with HSP27 (HSPB1) .

This methodology has been successfully used to identify HSPB8 protein interactions in various research contexts, including the characterization of the HSPB8-BAG3 complex involved in autophagy regulation .

How should I interpret discrepancies in HSPB8 expression patterns across different detection methods?

When facing discrepancies in HSPB8 expression detected by different methods, consider these methodological approaches to resolution:

Common discrepancies and resolution strategies:

• mRNA vs. protein level discrepancies:

  • In one study, researchers observed that estradiol and its valerate form increased both HSPB8 mRNA and protein levels, while tamoxifen increased only mRNA (two-fold) but not protein levels in MCF-7 cells .

  • Resolution approach: Consider post-transcriptional regulation mechanisms. Measure protein stability/half-life using cycloheximide chase assays or pulse-chase experiments.

• Western blot vs. IHC inconsistencies:

  • Resolution approach: Verify antibody specificity using knockdown controls. Consider tissue/cell heterogeneity in IHC that might be masked in whole-tissue Western blots.

• Variable detection across tissue types:

  • HSPB8 expression varies significantly across tissues, being highly expressed in heart, brain, and certain cancer tissues .

  • Resolution approach: Always include tissue-specific positive and negative controls for each detection method.

• Temporal expression differences:

  • HSPB8 expression can change dynamically over time. In the optic nerve crush model, temporal variations were observed .

  • Resolution approach: Design time-course experiments to capture expression dynamics.

• Antibody-specific variations:

  • Different antibodies may recognize different epitopes or be affected differently by post-translational modifications.

  • Resolution approach: Validate findings using multiple antibodies targeting different epitopes of HSPB8.

For publication-quality data, multiple detection methods should be employed to establish consensus on HSPB8 expression patterns. Where discrepancies persist, they should be explicitly discussed in relation to biological variables rather than dismissed.

What factors might affect the reproducibility of HSPB8 detection in experimental systems?

Several methodological factors can impact the reproducibility of HSPB8 detection:

Antibody-related factors:

• Antibody specificity: Different antibodies may recognize different epitopes or be affected by post-translational modifications of HSPB8.

  • Solution: Validate with multiple antibodies and include proper controls (e.g., HSPB8 knockdown samples).

• Lot-to-lot variability: Especially relevant for polyclonal antibodies.

  • Solution: Record lot numbers and test new lots against previous standards.

• Storage conditions: Antibody activity may decrease with improper storage.

  • Solution: Follow manufacturer recommendations. Generally, store at -20°C and avoid freeze-thaw cycles .

Sample preparation factors:

• Extraction buffer composition: Different buffers extract HSPB8 with varying efficiency. Some researchers found gel loading buffer more effective than RIPA buffer for zebrafish embryos .

  • Solution: Standardize extraction protocols and validate with positive controls.

• Post-translational modifications: Phosphorylation at Ser57 affects HSPB8 chaperone activity , which could alter antibody recognition.

  • Solution: Consider phospho-specific antibodies if phosphorylation is relevant.

Biological factors:

• Cell/tissue state: HSPB8 is a stress-responsive protein, and its expression can vary with cellular stress levels .

  • Solution: Standardize culture conditions and document any stressors.

• Cell cycle variations: HSPB8 expression and function may vary with cell cycle phase .

  • Solution: Consider cell synchronization for in vitro studies.

• Species differences: Despite 94% amino acid identity between human, mouse, and canine HSPB8 , species-specific differences in expression patterns exist.

  • Solution: Validate antibodies specifically for your species of interest.

To maximize reproducibility, detailed methodological documentation is essential, including antibody catalog numbers, dilutions, incubation times, and buffer compositions. Standardized positive controls should be included in each experimental run.

How does HSPB8 contribute to autophagy regulation, and what methodologies best capture this function?

HSPB8's role in autophagy regulation is complex and can be studied through several methodological approaches:

Molecular mechanisms of HSPB8 in autophagy:

• HSPB8-BAG3 complex formation:

  • HSPB8 forms a complex with BAG3 that regulates macroautophagy .

  • The complex recruits HSP70 and mediates chaperone-assisted selective autophagy .

• Autophagosome-lysosome fusion:

  • HSPB8 promotes the fusion of autophagosomes and lysosomes during autophagy .

  • The HSPB8 K141E mutation impairs this fusion process .

• Autophagosome formation:

  • Research suggests HSPB8 stimulates the forming process of autophagosomes rather than their degradation .

  • This is supported by observations that HSPB8 inhibition has similar effects as 3-methyladenine (3MA), which blocks autophagosome formation .

Methodological approaches to study HSPB8 in autophagy:

• Protein-protein interaction studies:

  • Co-immunoprecipitation to detect HSPB8-BAG3 complex formation .

  • Proximity ligation assays for in situ detection of protein interactions.

• Autophagy flux measurement:

  • Western blot analysis of autophagy markers (LC3-I/II, p62/SQSTM1) following HSPB8 manipulation.

  • Fluorescent reporters like GFP-LC3 or mRFP-GFP-LC3 to monitor autophagosome formation and maturation.

• Electron microscopy:

  • Transmission electron microscopy (TEM) to visualize autophagic structures .

  • Immunogold labeling to localize HSPB8 within autophagic structures.

• Pharmacological manipulations:

  • Combining HSPB8 knockdown with autophagy modulators (3MA as inhibitor, rapamycin as inducer) to determine pathway interactions .

  • Comparing HSPB8 knockdown phenotypes with known autophagy modulator effects.

• In vivo models:

  • AAV2-shHSPB8 has been used in mouse models to study HSPB8's role in autophagy and neuroprotection .

What are the current research approaches for studying HSPB8 in cancer progression?

Research on HSPB8 in cancer progression employs several sophisticated methodological approaches:

Expression analysis in patient samples:

• Tissue microarray analysis:

  • Immunohistochemical staining of cancer and adjacent normal tissues to evaluate HSPB8 expression patterns .

  • In bladder cancer studies, 68 cancer samples and 40 adjacent normal tissues were examined, revealing significantly elevated HSPB8 expression in cancer tissues .

• Public database mining:

  • Analysis of TCGA (The Cancer Genome Atlas) data for correlations between HSPB8 expression and clinical parameters .

  • The TCGA-BLCA (Bladder Urothelial Carcinoma) dataset has been used to categorize samples based on HSPB8 expression levels .

Functional studies in cancer models:

• Knockdown approaches:

  • siRNA-mediated silencing to evaluate the impact of HSPB8 reduction on cancer cell behavior .

  • In bladder cancer cells, HSPB8 knockdown reduced proliferation and migration while increasing apoptosis .

• Overexpression models:

  • Transfection with HSPB8 expression vectors to assess oncogenic potential .

  • In MCF-7 cells, HSPB8 overexpression resulted in minor changes in cell cycle distribution .

• Signaling pathway analysis:

  • Human Phospho-Kinase Array to identify downstream targets affected by HSPB8 manipulation .

  • In bladder cancer, HSPB8 knockdown decreased levels of eNOS, HSP27, PRAS40, RSK1/2, and STAT3 phosphorylation .

• In vivo tumor models:

  • Mouse xenograft models to evaluate the impact of HSPB8 manipulation on tumor growth in vivo .

Bioinformatic approaches:

• Gene set enrichment analysis (GSEA):

  • Identification of pathways and biological processes associated with HSPB8 expression .

  • Cancer-related pathways including cytokine-cytokine receptor interaction, focal adhesion, and proteoglycans in cancer have been identified .

• Protein-protein interaction (PPI) networks:

  • Construction of networks to visualize HSPB8 interactions in cancer contexts .

• Survival analysis:

  • Construction of survival prediction models based on HSPB8 expression levels .

  • HSPB8 has been investigated as a potential prognostic biomarker in bladder cancer .