BOLA2 Antibody

What is BOLA2 and what is its biological significance in research?

BOLA2 (BolA-Like Protein 2) is a member of the BolA protein family that plays a crucial role in intracellular iron homeostasis regulation . Research indicates that BOLA2 has significant oncological relevance, particularly in hepatocellular carcinoma (HCC), where its expression is markedly elevated compared to non-tumorous tissue . BOLA2 functions as a key regulatory protein for maintaining iron balance in the cellular microenvironment, and dysregulation of this homeostasis has been linked to cancer initiation and progression .

Pan-cancer analysis demonstrates that BOLA2 is overexpressed in multiple solid tumors beyond liver cancer, including breast, colorectal, and pancreatic cancers, suggesting its broad relevance as a research target . Functionally, BOLA2 has been shown to potently enhance c-Myc's oncogenic activity specifically in liver cancer, making it a compelling subject for mechanistic studies in oncology research .

What experimental applications are BOLA2 antibodies validated for?

BOLA2 antibodies have been validated for multiple experimental applications essential to cancer and molecular biology research:

When selecting an antibody for specific applications, researchers should consider:

• The cellular compartmentalization of BOLA2 being predominantly cytoplasmic

• The need for appropriate positive and negative controls specific to the application

• The sample type (cell lysates, tissue homogenates, or fixed specimens) when determining optimal antibody concentration

What are the optimal protocols for BOLA2 detection in tissue samples?

For immunohistochemical detection of BOLA2 in hepatocellular carcinoma and other tissue samples, researchers have successfully employed the following protocol components:

• Tissue fixation: Standard formalin fixation followed by paraffin embedding (FFPE) preserves BOLA2 epitopes well for detection

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes is recommended as BOLA2 epitopes can be masked by fixation processes

• Blocking: Use 3-5% normal serum corresponding to the secondary antibody host species for 1 hour at room temperature to minimize background staining

• Primary antibody: Rabbit polyclonal antibodies against human BOLA2 have demonstrated high specificity. Optimal dilution typically ranges from 1:100 to 1:500 depending on the specific antibody and tissue type

• Detection system: Either HRP-conjugated or biotin-conjugated secondary antibodies paired with appropriate visualization reagents have proven effective

• Counterstaining: Hematoxylin provides optimal nuclear contrast for evaluating BOLA2 cytoplasmic expression patterns

For quantification purposes, researchers have successfully used scoring systems combining staining intensity and percentage of positive cells to categorize samples into low and high BOLA2 expression groups .

How is BOLA2 expression effectively quantified at the mRNA level?

For quantitative evaluation of BOLA2 mRNA expression, researchers have successfully implemented qRT-PCR with the following methodological considerations:

• Primer design: The validated primer sequences for human BOLA2 amplification are:

  • Forward: 5'-CTGTAGCTTCCGAGTCCTG-3'

  • Reverse: 5'-TTCAAAGGCATGGATGTGC-3'

• Reference gene: β-actin has been validated as an appropriate housekeeping gene for BOLA2 expression studies, with primers:

  • Forward: 5'-GGACTTCGAGCAAGAGATGG-3'

  • Reverse: 5'-AGCACTGTGTTGGCGTACAG-3'

• Expression analysis: The 2^-ΔΔCt method is recommended for relative quantification, with expression values normalized to both reference genes and control samples

• Sample processing: RNA extraction should be performed with minimal delay after tissue collection to prevent degradation that could affect BOLA2 detection

In clinical research settings, BOLA2 mRNA measurements have successfully differentiated between HCC tumor tissues and non-tumorous liver tissues, with significant overexpression observed in tumorous samples (P<0.05) .

How do BOLA2 expression levels correlate with clinicopathological features in cancer research?

Research has identified significant correlations between BOLA2 expression and multiple clinicopathological features in hepatocellular carcinoma patients. The following data table summarizes these associations:

When designing studies investigating these correlations, researchers should implement a multivariate analysis approach using Cox regression models to control for confounding variables . This approach has confirmed BOLA2 as an independent prognostic factor in HCC (HR=2.108, 95% CI 1.541 to 6.067) .

What methodological approaches are optimal for investigating BOLA2's relationship with oxidative stress pathways?

BOLA2's involvement in oxidative stress regulation has been demonstrated through correlations with key regulatory proteins. When designing experiments to investigate these relationships, researchers should consider:

• Co-expression analysis: TIMER analysis has revealed positive correlations between BOLA2 mRNA expression and reactive oxygen species (ROS) modulator genes including:

  • p62 (positive correlation)

  • Autophagy-related 4B (ATG4B) (positive correlation)

  • Kelch-like ECH-associated protein 1 (Keap1) (positive correlation)

  • Inverse correlation with nuclear factor erythroid 2-related factor 2 (NRF2)

• Multiplexed immunohistochemistry: This approach enables simultaneous detection of BOLA2 and oxidative stress markers in the same tissue section, allowing direct visualization of spatial relationships

• Functional validation: CRISPR/Cas9-mediated knockout models of BOLA2 have successfully demonstrated causative relationships between BOLA2 and iron overload, which directly impacts ROS generation

• Pathway analysis: When examining BOLA2's role in redox regulation, focus on the p62-Keap1 signaling axis, as bioinformatic and immunohistochemical analyses confirm strong associations between BOLA2 and this pathway

Researchers should consider both direct effects (iron homeostasis disruption) and indirect mechanisms (modulation of autophagic processes through ATG4B) when designing comprehensive experiments to elucidate BOLA2's role in oxidative stress regulation.

What controls and validation steps are essential when using BOLA2 antibodies for immunohistochemistry?

To ensure reliable and reproducible results when using BOLA2 antibodies for immunohistochemistry, researchers should implement the following validation steps:

• Positive control selection: HCC tissues with confirmed high BOLA2 expression by orthogonal methods (e.g., qRT-PCR) serve as optimal positive controls . Additionally, other cancer types with established BOLA2 overexpression (breast, colorectal, pancreatic) can provide appropriate positive control tissues

• Negative controls:

  • Omission of primary antibody while maintaining all other steps

  • Use of non-specific IgG of the same isotype and concentration as the BOLA2 antibody

  • Normal liver tissues, which express significantly lower levels of BOLA2 compared to HCC, serve as biological negative controls

• Antibody validation:

  • Confirmation of specificity through Western blotting showing a single band at the expected molecular weight

  • Peptide competition assays to demonstrate binding specificity

  • Testing multiple antibody clones targeting different epitopes of BOLA2

• Technical considerations:

  • Standardize fixation time to minimize variability in epitope preservation

  • Include internal reference tissues within each staining batch

  • Implement digital image analysis for objective quantification of staining intensity and distribution

  • Validate scoring systems through independent assessment by multiple pathologists

• Correlation with other markers: Co-staining or sequential staining of BOLA2 with established markers like Ki-67 provides functional context, as research has shown significant correlations between BOLA2 expression and proliferation markers in HCC with tumor hemorrhage

How can researchers effectively study BOLA2's role in iron homeostasis using antibody-based approaches?

BOLA2's key function in iron homeostasis regulation requires specific experimental approaches when using antibody-based detection methods:

• Co-immunoprecipitation studies: BOLA2 antibodies can be used to pull down protein complexes involved in iron sensing and transport. Researchers should target:

  • GLRX3 (glutaredoxin 3), a known binding partner of BOLA2 in iron-sulfur cluster biogenesis

  • Iron regulatory proteins (IRPs) to assess potential regulatory interactions

  • Components of the cytosolic iron-sulfur assembly (CIA) machinery

• Subcellular localization analysis: Using immunofluorescence with BOLA2 antibodies to:

  • Track BOLA2 redistribution under iron-replete versus iron-depleted conditions

  • Co-localize BOLA2 with markers of specific subcellular compartments involved in iron metabolism

  • Examine potential shuttling between cytosolic and mitochondrial compartments

• Expression correlation studies: Combine BOLA2 antibody staining with iron-related markers:

  • Ferritin (iron storage protein)

  • Transferrin receptor (iron uptake)

  • Ferroportin (iron export)

  • Hepcidin (iron homeostasis regulator)

• Functional readouts: When assessing iron homeostasis disruption following BOLA2 manipulation, researchers should measure:

  • Labile iron pool using fluorescent indicators

  • Mitochondrial iron levels using specific dyes

  • ROS production as a functional consequence of iron dysregulation

• Validation in multiple models: BOLA2's effects on iron homeostasis should be confirmed across:

  • Multiple cell lines with varying baseline iron requirements

  • Patient-derived samples representing different disease stages

  • Animal models to capture systemic iron regulation effects

What experimental design considerations are important when studying BOLA2's role as a prognostic biomarker?

When investigating BOLA2 as a potential prognostic biomarker, particularly in HCC and other cancers, researchers should implement the following methodological approaches:

How can researchers address common technical challenges when using BOLA2 antibodies?

When encountering technical difficulties with BOLA2 antibody applications, researchers should implement the following troubleshooting approaches based on the specific issue:

• High background signal in immunohistochemistry:

  • Increase blocking duration (up to 2 hours) with 3-5% normal serum

  • Dilute primary antibody further (test series from 1:100 to 1:1000)

  • Include 0.1-0.3% Triton X-100 in blocking solutions to reduce non-specific binding

  • Optimize secondary antibody concentration independently of primary

• Weak or absent BOLA2 signal in positive control samples:

  • Evaluate epitope masking by testing multiple antigen retrieval methods (heat-induced vs enzymatic)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider signal amplification systems (tyramide signal amplification)

  • Verify antibody storage conditions to rule out degradation

• Inconsistent qRT-PCR results for BOLA2 mRNA detection:

  • Verify RNA integrity using electrophoresis or Bioanalyzer

  • Test multiple reference genes beyond β-actin

  • Optimize primer concentrations and annealing temperatures

  • Implement melt curve analysis to confirm amplification specificity

• Variability between different antibody lots:

  • Perform lot-to-lot validation using known positive controls

  • Maintain consistent antibody dilutions based on lot-specific titration

  • Consider pooling multiple antibody lots for long-term studies

  • Document lot numbers in experimental records

Researchers have successfully addressed these challenges by implementing standardized protocols and rigorous validation steps, resulting in reliable BOLA2 detection across multiple experimental platforms.

What experimental strategies can resolve contradictory findings in BOLA2 expression studies?

When research groups encounter contradictory results regarding BOLA2 expression or function, several methodological approaches can help resolve these discrepancies:

• Cross-validation with multiple detection methods:

  • Compare protein detection (IHC, Western blot) with mRNA analysis (qRT-PCR, RNA-seq)

  • Use multiple antibodies targeting different BOLA2 epitopes

  • Implement both chromogenic and fluorescent detection systems

  • Validate findings with orthogonal approaches like mass spectrometry

• Sample-specific considerations:

  • Stratify samples by disease stage, which can significantly impact BOLA2 expression patterns

  • Account for tumor heterogeneity by analyzing multiple regions from the same tumor

  • Control for treatment history, as interventions may alter BOLA2 expression

  • Consider ethnic and geographical differences in patient cohorts

• Technical standardization:

  • Implement uniform tissue processing protocols across research groups

  • Establish consensus cutoff values for defining "high" versus "low" expression

  • Use digital pathology for objective quantification

  • Share positive control samples between laboratories

• Functional validation:

  • Confirm phenotypic effects using both overexpression and knockdown/knockout models

  • Assess dose-dependent relationships rather than binary outcomes

  • Validate in multiple cell lines representing different tissue contexts

  • Implement rescue experiments to confirm specificity of observed effects

Recent studies highlighting BOLA2's role in tumor hemorrhage and prognosis in HCC employed this multi-faceted approach, demonstrating consistent findings across independent cohorts (n=96 and n=175) and multiple analytical methods (qRT-PCR, IHC, bioinformatic analysis) .