PDGFRB Antibody

What is PDGFRB and why is it a significant research target?

PDGFRB (platelet-derived growth factor receptor beta) is a 124 kDa receptor tyrosine kinase implicated in critical cellular processes. It forms dimers (homodimers α/α, β/β, or heterodimers α/β) upon ligand binding, triggering phosphorylation of downstream substrates. PDGFRB is expressed in embryonic tissues and mesenchymal-derived cells, playing significant roles in vascular development and pathological conditions including atherosclerosis and tumorigenesis . Its upregulation in most solid tumors, with expression in pericytes/smooth muscle cells, fibroblasts, macrophages, and certain tumor cells, makes it an important target for cancer research .

How do I select the appropriate anti-PDGFRB antibody for my experimental system?

Selection requires careful consideration of:

• Experimental application: Different antibodies perform optimally in specific applications (WB, IHC, FCM, etc.). For example, the APB5 clone has been validated for flow cytometry , while others like A00096-1 perform well in Western blot and IHC .

• Species reactivity: Determine which species your antibody recognizes. Some antibodies like IMC-2C5 bind both human and mouse PDGFRB with high affinity (0.014 and 0.061 nM respectively) , while others may be species-specific.

• Epitope specificity: For phosphorylation studies, phospho-specific antibodies targeting specific residues like Y740 are available .

• Validation data: Review published data showing antibody specificity in your application of interest. Look for validation images and assay conditions provided by manufacturers .

What are optimal conditions for detecting PDGFRB in Western blot applications?

For Western blot detection of PDGFRB:

• Sample preparation: Use 50μg of protein under reducing conditions from appropriate cell lines (e.g., HeLa, HepG2) or tissue lysates (e.g., testis tissue) .

• Gel parameters: Run samples on 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving) for 2-3 hours .

• Transfer conditions: Transfer to nitrocellulose membrane at 150mA for 50-90 minutes .

• Blocking and antibody incubation: Block with 5% non-fat milk/TBS for 1.5 hours at room temperature. Incubate with primary antibody at recommended dilution (0.5-2μg/mL) overnight at 4°C .

• Detection: Use appropriate secondary antibody and detection system (e.g., ECL kit) .

• Expected result: PDGFRB typically appears at approximately 160-180 kDa, slightly higher than the calculated 124 kDa due to post-translational modifications .

How can I optimize immunohistochemistry protocols for PDGFRB detection in tissue samples?

For optimal IHC detection:

• Antigen retrieval: Heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes is effective for many anti-PDGFRB antibodies, though some may require TE buffer pH 9.0 .

• Blocking: Block tissue sections with 10% goat serum to minimize non-specific binding .

• Antibody concentration: Start with 2μg/ml for rabbit anti-PDGFRB antibodies, or at manufacturer-recommended dilutions (typically 1:2000-1:8000) .

• Incubation conditions: Incubate primary antibody overnight at 4°C for optimal staining .

• Detection system: For indirect detection, biotinylated secondary antibodies followed by Streptavidin-Biotin-Complex (SABC) with DAB chromogen work effectively .

• Validated tissues: Placenta and kidney tissues have been validated for PDGFRB detection and can serve as positive controls .

How can I determine if my anti-PDGFRB antibody effectively blocks ligand-receptor interactions for functional studies?

To assess blocking capability:

• Binding inhibition assay: Mix various amounts of purified antibody with a fixed amount of PDGFRB (e.g., 50ng at 0.5μg/ml) and incubate (RT, 30 minutes). Transfer the mixture to plates precoated with PDGF-B (0.5μg/ml) and measure receptor binding. Calculate IC50 values to quantify blocking efficiency .

• Phosphorylation inhibition: Treat PDGFRB-expressing cells with the antibody prior to ligand stimulation. Use phospho-specific antibodies (e.g., against Y740) to determine if receptor activation is inhibited by Western blot .

• Downstream signaling assessment: Examine downstream signaling molecules like MAPK and Akt by Western blot to confirm functional blocking of pathway activation .

• Cell-based functional assays: Assess migration, proliferation, or survival of PDGFRB-expressing cells in the presence of blocking antibody and PDGF-B stimulation .

What strategies can I use to investigate PDGFRB's role in tumor angiogenesis and therapeutic resistance?

Several approaches have demonstrated efficacy:

• Combination therapy models: Combined targeting of PDGFRB and VEGFR2 pathways (e.g., using IMC-2C5 with DC101) has shown enhanced antitumor activity in multiple xenograft models (BxPC-3, NCI-H460, HCT-116) .

• Growth factor analysis: Analyze tumor homogenates by ELISA to assess how PDGFRB inhibition affects levels of other angiogenic factors like VEGF and bFGF .

• Resistance mechanism investigations: Monitor for emergence of mutations like PDGFRB C843G, which confers resistance to all generations of ABL TKIs including imatinib, dasatinib, nilotinib, and ponatinib .

• Alternative targeting approaches: For resistant mutations, evaluate alternative kinase inhibitors; for example, PDGFRB C843G mutant cells remain sensitive to multitarget kinase inhibitor CHZ868 .

• Clonal evolution tracking: Use longitudinal genomic profiling of samples collected during treatment to track emergence of resistance mechanisms .

What are common causes of variability in PDGFRB detection across different experimental systems?

Several factors can influence detection:

• Tissue-specific activation: PDGFRB activation may depend on microenvironment context. For example, PDGFR activation has been observed in tumor cells growing in bone but not in muscles, indicating context-dependent activation .

• PDGFRB expression heterogeneity: Expression levels can vary significantly across different cell lines and tissues, requiring optimization of antibody concentration for each system .

• Post-translational modifications: These affect molecular weight observed in Western blots (160-180 kDa observed vs. 124 kDa calculated), which may vary between cell types and activation states .

• Non-specific binding: Some antibodies may cross-react with PDGFRα; validation of specificity is essential .

• Antibody titration: Careful titration is required for optimal performance in different applications, with concentrations ranging from 0.5μg to 4.0μg depending on the experimental system .

How can I verify specificity when working with phospho-specific PDGFRB antibodies?

To ensure phospho-antibody specificity:

• Stimulation controls: Include unstimulated controls alongside PDGF-B-stimulated samples to confirm specificity for the phosphorylated form .

• Phosphatase treatment: Treat parallel samples with phosphatase to confirm the signal is phosphorylation-dependent.

• Mutant constructs: Generate tyrosine-to-phenylalanine mutants at specific phosphorylation sites (e.g., Y740F) as negative controls .

• Competing phosphopeptides: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides to demonstrate phospho-specificity.

• Kinase inhibitor treatment: Pre-treat cells with specific PDGFRB kinase inhibitors to show signal reduction in Western blots using phospho-specific antibodies .

How can anti-PDGFRB antibodies be used to investigate therapeutic resistance mechanisms in cancer?

Anti-PDGFRB antibodies enable several approaches:

• Mutation detection: Use antibodies to pull down PDGFRB protein for sequencing to identify resistance mutations, such as the PDGFRB C843G mutation observed in Ph-like ALL resistant to TKIs .

• Combinatorial efficacy testing: Evaluate anti-PDGFRB antibodies in combination with chemotherapy and other targeted therapies in resistant models. IMC-2C5 has shown additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models .

• Downstream signaling evaluation: Investigate alternative signaling pathways activated in resistant cells using phospho-specific antibodies against downstream targets .

• Immunohistochemical analysis: Use anti-PDGFRB antibodies to evaluate receptor expression and localization changes in resistant tumors .

• Novel fusion detection: Identify and characterize new oncogenic fusion genes involving PDGFRB, such as AGGF1-PDGFRB identified in Ph-like ALL, which may have different resistance profiles .

What methodological considerations are important when using anti-PDGFRB antibodies in translational oncology research?

Key considerations include:

• Species cross-reactivity: Antibodies with cross-reactivity between human and mouse PDGFRB (like IMC-2C5) enable assessment of both tumor cell and stromal cell-expressed receptor in xenograft models, more closely mirroring clinical scenarios .

• Expression-efficacy correlation analysis: There is often no direct correlation between level of PDGFRB expression on tumor cells and antibody efficacy, suggesting more complex mechanisms of action .

• Microenvironment consideration: PDGFRB activation depends on tumor-host microenvironment interactions, requiring careful experimental design to recapitulate these conditions .

• Quantitative receptor binding assays: Determine antibody affinities using surface plasmon resonance. For reference, IMC-2C5 binds hPDGFRβ and mPDGFRβ with affinities of 0.014 and 0.061 nM respectively .

• Translational biomarker development: Establish protocols to monitor PDGFRB expression/activation as potential biomarkers for patient stratification in clinical studies .