FBLN2 Antibody, HRP conjugated

What is Fibulin-2 (FBLN2) and why is it important in extracellular matrix research?

Fibulin-2 (FBLN2) is a secreted extracellular matrix glycoprotein critically involved in tissue development and remodeling processes . The protein plays significant roles in basement membrane integrity, particularly at the myoepithelial cell/basement membrane interface in normal mammary tissue . FBLN2 functions as an adapter that mediates interactions between fibrillin-1 (FBN1) and elastin (ELN) . Its binding to fibronectin and various other ligands is calcium-dependent, highlighting its importance in maintaining extracellular matrix structure and function .

Research indicates that FBLN2 is strongly associated with specific developmental stages, particularly during epithelial growth initiation and primary expansion in mammary gland development . The protein shows distinct temporal expression patterns, with highest abundance during pubertal and early pregnant stages, after which expression steadily decreases .

What are the optimal storage conditions for FBLN2 Antibody, HRP conjugated?

FBLN2 Antibody, HRP conjugated requires precise storage conditions to maintain functional integrity. According to manufacturer specifications, the antibody should be shipped at 4°C, and upon receipt, researchers should store it at -20°C for short-term storage or at -80°C for long-term storage . Multiple freeze-thaw cycles should be strictly avoided as they can compromise antibody activity and specificity .

The antibody is typically formulated in a specialized buffer containing 0.03% Proclin 300 (a preservative), 50% Glycerol, and 0.01M PBS at pH 7.4, designed to maintain stability during storage . This formulation helps preserve both the antibody structure and the conjugated HRP enzyme activity.

How do FBLN2 expression levels compare between normal tissues and cancer tissues?

FBLN2 expression shows significant variation between normal and cancerous tissues, with distinctive patterns in different cancer types. In hepatocellular carcinoma (HCC), analysis using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database revealed that FBLN2 is upregulated compared to normal liver samples . Immunohistochemical studies on HCC patient samples confirmed higher expression of FBLN2 in tumor tissues versus adjacent non-cancerous tissues .

Contrastingly, in breast cancer, strong FBLN2 staining is observed around normal ducts at the myoepithelial cell/basement membrane interface in morphologically normal breast tissue . The expression pattern changes during cancer progression, reflecting FBLN2's complex role in different malignancies.

The following table summarizes recovery rates when detecting FBLN2 in different matrices:

These findings demonstrate consistent detection capabilities across different biological matrices, supporting the reliability of FBLN2 antibodies in various research applications .

How does FBLN2 differentially influence cancer progression across various tumor types?

FBLN2 exhibits remarkable context-dependent functions across different cancer types, presenting an intriguing paradox for cancer researchers. In hepatocellular carcinoma (HCC), evidence suggests that FBLN2 facilitates malignant progression . Researchers have documented upregulated expression in HCC tissues compared to normal liver samples, suggesting its potential role as an oncogenic driver in this context .

Conversely, in breast cancer, FBLN2 demonstrates a more complex role. The protein appears to contribute to basement membrane integrity, potentially functioning as a barrier against invasion . High levels of FBLN2 mRNA are significantly associated with improved distant metastasis-free survival in patients with negative lymph node status (P = 0.03; n = 988) and in those with intermediate grade breast cancer (Grade II, P = 0.05; n = 546) . Intriguingly, this correlation reverses in high-grade breast cancer (Grade III), where higher FBLN2 mRNA levels associate with poorer outcomes (P = 0.023; n = 458) .

In lung adenocarcinoma, studies indicate that FBLN2 contributes to metastasis and invasion, aligning more with its role in HCC than in early-stage breast cancer . These contradictory findings across different cancer types highlight the need for context-specific research approaches when investigating FBLN2's role in oncology.

What methodological considerations are critical when using FBLN2 antibodies for immunohistochemistry in formalin-fixed tissues?

When performing immunohistochemistry with FBLN2 antibodies on formalin-fixed, paraffin-embedded tissues, several methodological considerations are essential for obtaining reliable and reproducible results:

For human endometrial carcinoma samples, researchers have reported successful staining using FBLN2 antibody diluted at 1:500 following citrate buffer antigen retrieval at pH 6.0 for 15 minutes .

How can researchers reconcile contradictory findings regarding FBLN2's role in tumor progression?

Reconciling contradictory findings regarding FBLN2's role in tumor progression requires comprehensive methodological approaches that address several research dimensions:

• Tissue context specificity: Researchers should recognize that FBLN2 functions differently across tissue types. Studies show FBLN2 promotes malignancy in hepatocellular carcinoma and lung adenocarcinoma while potentially suppressing progression in early-stage breast cancer . These findings suggest experiments should be designed with tissue-specific controls and comparisons rather than generalizing across cancer types.

• Disease stage stratification: Evidence indicates FBLN2's role changes during disease progression. In breast cancer, high FBLN2 mRNA levels correlate with better prognosis in early stages but worse outcomes in advanced disease . Experimental designs should therefore stratify samples by disease stage, grade, and molecular subtype to detect stage-dependent effects.

• Molecular pathway analysis: To understand mechanistic discrepancies, researchers should investigate FBLN2's interaction with tissue-specific signaling pathways. In HCC, preliminary exploration of possible mechanisms has revealed potential interactions with pathways affecting malignant progression . Comprehensive protein-protein interaction studies and pathway analyses across different tissue contexts can help explain divergent functions.

• Model system validation: Researchers should validate findings across multiple model systems (cell lines, patient-derived xenografts, and clinical samples) to ensure observations aren't artifacts of particular experimental systems. This multi-model approach helps distinguish genuine biological complexity from technical variability.

• Functional domain analysis: FBLN2's varied effects could result from differential expression of specific functional domains or isoforms across tissues. Detailed molecular characterization using domain-specific antibodies or isoform-specific detection methods can provide clarity on these mechanisms.

What are the recommended dilution ranges for FBLN2 Antibody, HRP conjugated across different experimental applications?

The effectiveness of FBLN2 Antibody, HRP conjugated varies significantly across experimental applications, requiring careful optimization of dilution factors. Based on empirical evidence and manufacturer recommendations, the following application-specific dilution ranges have been established:

For Western blot applications, whole cell extracts (30 μg) separated by 5% SDS-PAGE have been successfully detected using FBLN2 antibody diluted at 1:500 . The detection is typically performed using an HRP-conjugated anti-rabbit IgG antibody as the secondary detection reagent .

For immunohistochemistry on paraffin-embedded tissues, successful staining has been reported at 1:500 dilution for human endometrial carcinoma samples . Optimization for each specific tissue type and fixation method is recommended, as antigen accessibility can vary considerably between different sample preparations.

How can researchers effectively validate the specificity of FBLN2 antibodies in their experimental systems?

Validating the specificity of FBLN2 antibodies requires a multi-faceted approach to ensure reliable experimental outcomes:

• Positive and negative control tissues: Include tissues known to express high levels of FBLN2 (such as pubertal mammary glands and early pregnant mouse mammary epithelium ) as positive controls. Tissues with minimal FBLN2 expression or FBLN2 knockout models serve as negative controls.

• Antibody validation in FBLN2 knockdown/knockout systems: Utilize FBLN2 stable knockdown cell lines transduced with lentiviral vectors (such as those targeting sequences 5′-GCTGCACCACGGAGAGTTTCA-3′ and 5′-GCAACTGTGTGGACATCAACG-3′) to confirm antibody specificity. The antibody signal should be substantially reduced in knockdown/knockout systems.

• Western blot verification: Confirm antibody specificity by Western blot, looking for the expected ~195 kDa band that corresponds to FBLN2 protein . Multiple tissue samples should be tested to verify consistent binding specificity.

• Cross-reactivity assessment: While available assays show high sensitivity and excellent specificity for detection of FBLN2 with no significant cross-reactivity or interference between FBLN2 and analogues , researchers should independently verify this specificity in their specific experimental context.

• mRNA correlation: Correlate protein expression detected by the antibody with mRNA expression levels measured by qRT-PCR using primers such as FBLN2-F:5′-CTGCTACAAGGCACTCACCTGT-3′ and FBLN2-R:5′-GTAGAAGGAGCCCTTGGTGTTC-3′ . Concordance between protein and mRNA levels supports antibody specificity.

• Competition assays: Perform pre-absorption of the antibody with purified recombinant FBLN2 protein (such as recombinant Human Fibulin-2 protein covering amino acids 301-440) prior to application in the experimental system. Disappearance of signal confirms specific binding.

What experimental considerations are important when investigating FBLN2 expression in cancer progression models?

When investigating FBLN2 expression in cancer progression models, researchers should address several critical experimental considerations:

• Comprehensive sampling across tumor progression stages: FBLN2 expression changes during cancer progression, with different implications in early versus advanced disease stages. In breast cancer studies, researchers should include morphologically normal breast tissue, ductal carcinoma in situ (DCIS), and invasive cancer samples to track expression changes across the progression continuum .

• Stromal versus epithelial expression analysis: FBLN2 localizes differently in tissue compartments, showing strong staining around normal ducts at the myoepithelial cell/basement membrane interface and in interlobular stroma . Microdissection techniques or dual immunofluorescence staining should be employed to distinguish compartment-specific expression patterns.

• Correlation with basement membrane integrity markers: Given FBLN2's role in basement membrane integrity, co-staining with basement membrane markers provides crucial contextual information. Researchers have used smooth muscle actin (SMA) and vimentin staining alongside FBLN2 to establish its relationship to the myoepithelial cell/basement membrane interface .

• Standardized quantification methods: For immunohistochemical studies, employ standardized scoring systems such as the Quickscore method (combines percent-positive staining with intensity scoring), as shown in this example table:

• Multi-parametric analysis: Correlate FBLN2 expression with clinical parameters such as lymph node status, tumor grade, and patient survival. Research has shown that the prognostic value of FBLN2 mRNA levels varies significantly between lymph node negative patients (P = 0.03; n = 988) and those with different tumor grades .

What are common pitfalls when performing Western blot analysis with FBLN2 antibodies and how can they be addressed?

When performing Western blot analysis with FBLN2 antibodies, researchers frequently encounter several technical challenges that can be systematically addressed:

• Poor detection of high molecular weight FBLN2 band (~195 kDa):

  • Problem: FBLN2 is a large glycoprotein that may transfer inefficiently from gel to membrane.

  • Solution: Use lower percentage SDS-PAGE gels (5%) for better resolution of high molecular weight proteins . Extend transfer time or use specialized transfer systems designed for high molecular weight proteins. Consider wet transfer methods with low methanol buffers.

• Multiple bands or non-specific binding:

  • Problem: Multiple bands may represent different FBLN2 isoforms, proteolytic fragments, or non-specific binding.

  • Solution: Optimize blocking conditions (5% non-fat dry milk or bovine serum albumin). Increase antibody dilution (try 1:1000-1:3000 range) . Verify specificity using FBLN2 knockdown samples as negative controls. Consider using FBLN2 antibodies validated specifically for Western blot applications.

• Weak or absent signal:

  • Problem: Low abundance of FBLN2 in certain samples or protein degradation.

  • Solution: Load adequate protein (minimum 30 μg of whole cell extracts) . Include protease inhibitors during sample preparation. Confirm protein transfer efficiency with reversible membrane staining. Consider enriching for extracellular matrix proteins during sample preparation.

• High background:

  • Problem: Non-specific binding of primary or secondary antibodies.

  • Solution: Increase washing duration and frequency with TBST. Optimize antibody dilutions and blocking conditions. Reduce antibody incubation time or consider switching to a more specific secondary antibody.

• Inconsistent results across different tissue/cell types:

  • Problem: Varying FBLN2 expression levels and post-translational modifications.

  • Solution: Include positive control samples known to express FBLN2 (such as pubertal mammary gland tissue) . Normalize loading using appropriate housekeeping proteins such as β-actin or GAPDH .

How can researchers optimize ELISA protocols for reliable quantification of FBLN2 in different biological matrices?

Optimizing ELISA protocols for FBLN2 quantification requires meticulous attention to several critical parameters:

• Matrix-specific validation: Different biological matrices affect FBLN2 detection differently. Recovery rates vary between serum (91-102%, average 93%), EDTA plasma (87-100%, average 91%), and heparin plasma (90-101%, average 96%) . Each matrix should be validated independently, and matrix-matched calibration curves should be prepared when possible.

• Sample preparation standardization: Standardize pre-analytical variables including collection tubes, processing time, centrifugation parameters, and storage conditions. For plasma samples, compare EDTA and heparin anticoagulants to determine optimal collection method for your specific application .

• Dilution optimization: Perform preliminary experiments with serial dilutions of samples to identify the optimal dilution factor that places readings within the linear range of the standard curve. This is particularly important for FBLN2, which may be present at varying concentrations across different tissue types and disease states.

• Antibody pair selection: For sandwich ELISA, the capture antibody pre-coated onto the 96-well plate and the biotin-conjugated detection antibody must recognize discrete, non-overlapping epitopes on FBLN2 . HRP-conjugated FBLN2 antibodies may be particularly suitable for competitive ELISA formats .

• Standard curve optimization: Prepare a fresh standard curve for each assay using recombinant FBLN2 protein. Ensure adequate points in the low concentration range for sensitive detection. Log-log transformation of standard curve data often improves curve fitting for immunoassays.

• Signal amplification considerations: For low-abundance samples, consider signal amplification strategies such as extended substrate incubation times or amplification systems compatible with HRP-conjugated antibodies .

• Cross-reactivity assessment: Although existing assays demonstrate high specificity with no significant cross-reactivity between FBLN2 and analogues , researchers should independently verify specificity in their experimental system, particularly when analyzing complex biological matrices.

What approaches can resolve contradictory data when analyzing FBLN2 expression across different experimental platforms?

When researchers encounter contradictory data regarding FBLN2 expression across different experimental platforms, several methodological approaches can help resolve these discrepancies:

• Multi-platform validation protocol: Implement a systematic validation strategy that examines FBLN2 at both mRNA and protein levels. This should include qRT-PCR using validated primer pairs (such as FBLN2-F:5′-CTGCTACAAGGCACTCACCTGT-3′ and FBLN2-R:5′-GTAGAAGGAGCCCTTGGTGTTC-3′) , Western blot analysis, immunohistochemistry, and when possible, mass spectrometry-based proteomics.

• Reference gene/protein normalization assessment: Carefully evaluate normalization strategies for each platform. For qRT-PCR, researchers have used different reference genes including GAPDH and keratin markers (Krt18, Krt14) to normalize FBLN2 expression. Different normalization strategies can yield apparently contradictory results - for example, research has shown that when normalized to Krt18, FBLN2 mRNA appears abundant in both pubertal and early pregnant mice, but when normalized to Krt14, the strongest expression is only found during puberty .

• Spatial resolution analysis: Implement techniques that provide spatial context to expression data. In pubertal mammary glands, FBLN2 localizes predominantly to terminal end buds (TEBs) rather than ducts . Whole-tissue analysis may obscure these important spatial differences. Compare isolated TEB and ductal tissue, or use techniques like laser capture microdissection to achieve appropriate spatial resolution.

• Isoform-specific detection strategies: Design experiments that can distinguish between potential FBLN2 isoforms or post-translationally modified forms. Western blot analysis has identified a major ~195 kDa band in mammary tissues , but other isoforms may exist and be detected differently across platforms.

• Temporal dynamics consideration: FBLN2 expression varies significantly across developmental stages. In mammary gland development, expression is highest during puberty and early pregnancy, then steadily decreases . Ensure sampling timepoints are precisely matched when comparing across platforms to avoid temporal artifacts.

• Controlled microenvironment experiments: When possible, use controlled in vitro systems to verify expression patterns observed in complex tissues. This allows manipulation of specific variables that might influence FBLN2 expression while maintaining consistent experimental conditions across different analytical platforms.