HDLBP Antibody

What is HDLBP and what are its primary biological functions?

HDLBP is the human member of the vigilin protein family characterized by 14-15 RNA-interacting KH (heterologous nuclear ribonucleoprotein K homology) domains. It primarily localizes to the cytoplasmic face of the endoplasmic reticulum but can also be found in the cytosol and nucleus. HDLBP is involved in multiple cellular processes including translation, chromosome segregation, cholesterol transport, and carcinogenesis . The protein binds high-density lipoprotein (HDL) and may function to regulate excess cholesterol levels in cells. Additionally, it binds RNA and can induce heterochromatin formation . Three transcript variants encoding two different isoforms have been identified for this gene .

What types of HDLBP antibodies are available for research?

Researchers can access various types of HDLBP antibodies for experimental applications. These include polyclonal antibodies, such as rabbit-derived polyclonal antibodies that recognize human, mouse, and rat HDLBP . These antibodies can be used in multiple applications including ELISA, immunohistochemistry (IHC), and Western blotting (WB) . ELISA kits with high sensitivity and specificity for HDLBP detection are also commercially available for quantitative measurement in human tissue homogenates, cell lysates, and cell culture supernatants .

What are the optimal methods for detecting HDLBP in different sample types?

The detection of HDLBP can be accomplished through several complementary techniques, depending on the research question and sample type:

Western Blotting: Effective for quantifying HDLBP protein levels in cell lysates and tissue homogenates. Typically requires optimization of protein extraction protocols to maximize yield while preserving protein integrity. Antibodies like the HDLBP polyclonal antibody (E-AB-16475) are suitable for this application .

Immunohistochemistry (IHC): Valuable for visualizing HDLBP localization within tissue sections. This approach has been used to demonstrate the shift of HDLBP from nuclear to cytoplasmic localization in breast cancer progression .

ELISA: Provides quantitative measurement of HDLBP with detection ranges of approximately 0.31-20 ng/mL and detection limits as low as 0.138 ng/mL . Sandwich ELISA methods offer high sensitivity and excellent specificity for HDLBP detection in various sample types .

How can researchers validate HDLBP antibody specificity?

Validating antibody specificity is crucial for obtaining reliable experimental results. Recommended validation approaches include:

• Positive and negative control samples: Using samples with known HDLBP expression levels and samples where HDLBP has been knocked down.

• Western blot analysis: Confirming single band detection at the expected molecular weight (approximately 110-160 kDa for full-length HDLBP) .

• Peptide competition assays: Pre-incubating the antibody with the immunogen peptide should abolish specific signal.

• Cross-reactivity testing: Evaluating the antibody against related proteins to ensure specificity.

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of HDLBP to confirm consistent results.

What considerations are important when using HDLBP antibodies for immunohistochemistry?

When conducting immunohistochemistry with HDLBP antibodies, researchers should consider:

• Fixation protocols: Optimal fixation varies by tissue type; overfixation can mask epitopes.

• Antigen retrieval: Often necessary to expose epitopes masked by fixation.

• Background reduction: Use appropriate blocking solutions to minimize non-specific binding.

• Antibody dilution optimization: Titration experiments to determine optimal antibody concentration.

• Appropriate controls: Include positive control tissues known to express HDLBP and negative controls (no primary antibody).

Immunohistochemical approaches have successfully demonstrated HDLBP's altered expression and localization in cancer progression, particularly in breast cancer where HDLBP shows decreased abundance in atypical ductal hyperplasia and a shift from nuclear to cytoplasmic localization in ductal carcinoma in situ .

How can HDLBP antibodies be used to study cancer progression mechanisms?

HDLBP antibodies serve as valuable tools for investigating cancer progression mechanisms:

• Epithelial-mesenchymal transition (EMT) analysis: HDLBP promotes EMT in hepatocellular carcinoma through B-RAF-dependent mechanisms. Antibodies can track HDLBP expression changes during this process .

• Cell migration studies: In vitro experiments using HDLBP antibodies can monitor protein expression in relation to migration capacity. Knockdown and overexpression studies have demonstrated HDLBP's role in promoting cell migration in hepatic cell lines .

• Tumor biomarker evaluation: HDLBP antibodies can be used to assess protein levels across cancer stages, as demonstrated in breast cancer where HDLBP abundance progressively decreases compared to normal tissue .

• Cancer type differentiation: Immunostaining with HDLBP antibodies shows potential for differentiating between malignant mesothelioma and lung adenocarcinoma, addressing a significant diagnostic challenge .

What approaches can be used to study HDLBP's interaction with specific mRNAs?

Understanding HDLBP's RNA-binding properties requires specialized techniques:

• RNA immunoprecipitation (RIP): Using HDLBP antibodies to pull down protein-RNA complexes, followed by RNA sequencing to identify bound transcripts.

• Cross-linking immunoprecipitation (CLIP): Providing higher resolution of RNA-protein interaction sites.

• Proximity labeling: Combining with RNA capture to identify RNAs in close proximity to HDLBP.

• Subcellular fractionation: Combined with HDLBP immunoprecipitation to study compartment-specific RNA interactions.

Research has shown that HDLBP primarily binds to mRNAs encoding secreted proteins and those localized to the ER membrane . These interactions are likely mediated through the protein's multiple KH domains.

How does HDLBP contribute to lipid metabolism and what tools can be used to study this function?

HDLBP's role in lipid metabolism can be investigated using:

• Cholesterol transport assays: Measuring HDL binding and cholesterol uptake in cells with modulated HDLBP levels.

• Lipoprotein profiling: Analyzing changes in lipoprotein composition and distribution in response to HDLBP manipulation.

• Co-immunoprecipitation studies: Using HDLBP antibodies to identify interactions with components of lipid metabolism pathways.

• Metabolic labeling: Tracking lipid synthesis and transport in cells with altered HDLBP expression.

The connection between HDLBP and cholesterol metabolism is further evidenced by the interaction of a processed 110kDa version of HDLBP with aralin (a plant-derived ribosome-inactivating protein), which is correlated with cellular cholesterol levels .

What are the common difficulties in studying HDLBP expression patterns?

Researchers face several challenges when investigating HDLBP expression:

• Isoform diversity: Multiple transcript variants encoding different isoforms complicate expression analysis .

• Post-translational modifications: Processed forms of HDLBP (e.g., the 110kDa version) may have distinct functions from the full-length protein .

• Subcellular localization shifts: HDLBP can relocate between cellular compartments, requiring comprehensive fractionation and imaging approaches.

• Context-dependent expression: HDLBP levels and functions vary significantly across tissue types and disease states.

To address these challenges, researchers should employ multiple detection methods, use antibodies that can distinguish between isoforms when possible, and carefully design experiments that account for HDLBP's complex biology.

How can researchers investigate HDLBP's role in drug resistance mechanisms?

HDLBP has been implicated in treatment resistance, particularly in hepatocellular carcinoma. Approaches to study this include:

• Drug sensitivity assays: Comparing drug responses in cells with normal, elevated, or reduced HDLBP levels.

• Signaling pathway analysis: HDLBP promotes RAF1 expression and regulates MEKK1 kinase activity, activating the RAF1-MAPK signaling pathway and contributing to sorafenib resistance .

• Protein stability studies: HDLBP competes with the E3 ubiquitin-protein ligase TRIM71 for binding to RAF1, inhibiting its degradation .

• Ferroptosis assays: HDLBP counters ferroptosis by stabilizing lncFAL, which restricts the effects of ferroptosis-inducing reagents .

What techniques can be used to study the domain-specific functions of HDLBP?

HDLBP's 15 KH domains likely confer distinct functionalities. To investigate domain-specific functions:

• Domain deletion/mutation constructs: Creating variants lacking specific KH domains to assess their contribution to different functions.

• Domain-specific antibodies: Developing antibodies that target individual domains to study their interactions and accessibility.

• Structural analysis: Using techniques like X-ray crystallography or cryo-EM to understand domain architecture.

• Interaction mapping: Identifying which domains are responsible for specific protein-protein interactions.

Research has shown that the C-terminal KH domains (13 and 14) are crucial for interaction with RACK1 and subsequent support of flaviviral replication . Similarly, the N-terminal region containing six KH domains mediates interaction with the tumor suppressor TSC2 .

How might HDLBP antibodies contribute to cancer diagnostics?

The potential for HDLBP as a diagnostic marker is promising:

• Early cancer detection: HDLBP expression changes have been detected even in early stages of breast cancer development such as atypical ductal hyperplasia .

• Cancer subtype differentiation: HDLBP immunostaining shows potential for differentiating between difficult-to-distinguish cancer types such as malignant mesothelioma and lung adenocarcinoma .

• Prognostic biomarker development: Higher HDLBP levels correlate with lower survival rates in hepatocellular carcinoma with vascular invasion .

• Treatment response prediction: HDLBP's role in sorafenib resistance suggests potential utility in predicting treatment outcomes .

The development of standardized immunohistochemical protocols using validated HDLBP antibodies could lead to clinical applications in cancer diagnostics.

What emerging techniques might enhance the study of HDLBP biology?

Advances in research methodologies offer new opportunities for HDLBP research:

• Single-cell analysis: Revealing cell-to-cell variation in HDLBP expression and function.

• CRISPR-based approaches: Enabling precise genome editing to study HDLBP function.

• Proximity labeling proteomics: Identifying context-specific HDLBP interaction partners.

• Advanced imaging techniques: Super-resolution microscopy to visualize HDLBP localization and dynamics.

• Patient-derived organoids: Providing more physiologically relevant models to study HDLBP in disease contexts.

How can researchers target specific functions of HDLBP for therapeutic development?

Targeted approaches to modulate HDLBP function include:

Research suggests that targeting specific regions or domains of HDLBP, rather than the entire protein, may offer more precise therapeutic approaches with fewer unintended consequences .