FOLR2 Antibody, HRP conjugated

What is FOLR2 and why is it a significant research target?

FOLR2 (Folate Receptor 2), also known as Folate Receptor beta, is a 38 kDa glycoprotein anchored to the cell membrane via glycosyl phosphatidylinositol (GPI) linkage. It mediates cellular uptake of folic acid and reduced folates, which are required for key metabolic processes including nucleotide synthesis, methionine synthesis, and amino acid metabolism . FOLR2 is predominantly expressed in placenta, myeloid cells, and some CD34+ hematopoietic progenitor cells . It has emerged as an important research target because:

• It is upregulated in myeloid leukemias, head and neck squamous cell carcinomas, and several non-epithelial cancers

• It shows increased expression on macrophages and monocytes at chronic inflammatory sites, including rheumatoid arthritis synovium and glioblastoma

• It has been implicated in cancer cell proliferation pathways

• Its expression pattern makes it a potential diagnostic marker and therapeutic target

What are the key differences between detection methods for FOLR2 protein?

Different detection methods offer complementary information about FOLR2 expression and localization:

For Western blot detection of FOLR2, the primary antibody is typically followed by an HRP-conjugated secondary antibody to visualize the protein band at approximately 38 kDa under reducing conditions .

How should researchers optimize antibody dilutions for FOLR2 detection in Western blot applications?

Optimization of antibody dilutions is critical for specific FOLR2 detection while minimizing background:

• Start with manufacturer's recommended dilution. For example, Human FOLR2 Antigen Affinity-purified Polyclonal Antibody (AF5697) is typically used at 1 μg/mL , while other FOLR2 antibodies may be recommended at 1:1000-1:2000 dilutions .

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) of the primary FOLR2 antibody to identify optimal signal-to-noise ratio.

• Similarly optimize the HRP-conjugated secondary antibody dilution (typically starting at 1:2000-1:5000).

• Include appropriate controls:

  • Positive control (known FOLR2-expressing tissue/cells like placental tissue or NCI-H1650 cells)

  • Negative control (cells with low FOLR2 expression like normal HBE cells)

  • Secondary antibody-only control to assess non-specific binding

• Optimize blocking conditions (typically 5% non-fat milk or BSA) and washing steps to reduce background.

The optimal antibody concentration provides a clear specific band at 38 kDa with minimal background and no non-specific bands.

What sample preparation methods best preserve FOLR2 epitopes for antibody detection?

Proper sample preparation is crucial for maintaining FOLR2 antigenicity:

• Cell/Tissue Lysis:

  • Use RIPA buffer or dedicated immunoblot buffer (e.g., Immunoblot Buffer Group 8) with protease inhibitors.

  • Maintain samples at 4°C during processing to prevent degradation.

  • For GPI-anchored proteins like FOLR2, include detergents that effectively solubilize membrane proteins.

• Fixation for Immunocytochemistry:

  • For cellular localization studies, immersion fixation has been successfully used for FOLR2 detection in neutrophils .

  • For flow cytometry applications, gentle fixation preserves the native epitope conformation.

• Protein Quantification:

  • Use BCA or Bradford assay to ensure equal loading of protein for comparative studies.

  • Standard loading is typically 20-50 μg of total protein per lane for Western blot.

• Denaturation Conditions:

  • FOLR2 detection has been successfully performed under reducing conditions , but optimization may be required for specific antibodies.

What are common causes of weak or absent FOLR2 signal in Western blot, and how can they be addressed?

When FOLR2 signal is weak or absent despite expecting expression, consider these issues and solutions:

FOLR2 typically appears as a band at approximately 38 kDa, though glycosylation may cause slight size variations .

How can researchers differentiate between FOLR1, FOLR2, and FOLR3 when using antibodies?

Distinguishing between folate receptor family members requires careful antibody selection and experimental design:

• Antibody Selection:

  • Choose antibodies validated for specificity against FOLR2 with minimal cross-reactivity to FOLR1 and FOLR3.

  • Review cross-reactivity data in antibody datasheets; FOLR2 has 68% homology with FOLR1 and 79% with FOLR3 .

• Expression Pattern Analysis:

  • FOLR1 is predominantly expressed in epithelial tissues

  • FOLR2 is expressed in placenta, myeloid cells, and some CD34+ hematopoietic progenitor cells

  • FOLR3 is expressed primarily in hematopoietic tissues

• Molecular Weight Discrimination:

  • Although similar in size, careful SDS-PAGE resolution can help distinguish the different folate receptors

  • Use positive control lysates for each receptor to establish correct band positions

• Validation Approaches:

  • siRNA knockdown of FOLR2 can confirm antibody specificity

  • Recombinant protein controls for each receptor can serve as specificity controls

  • Peptide competition assays using receptor-specific peptides

How can FOLR2 antibodies be effectively used to study M2 macrophage polarization in tumor microenvironments?

FOLR2 is increasingly recognized as a marker of M2-polarized macrophages in tumor microenvironments. For such studies:

• Multi-parameter Flow Cytometry:

  • Combine FOLR2 antibodies with other M2 markers (CD163, CD206) and general macrophage markers (CD68, CD11b)

  • Use an antibody panel: FOLR2-PE + CD163-APC + CD206-FITC + CD68-PerCP-Cy5.5 for comprehensive phenotyping

  • Include appropriate isotype controls for each fluorochrome

• Immunohistochemistry of Tumor Sections:

  • Dual staining with FOLR2 and other macrophage markers can identify M2-polarized tumor-associated macrophages

  • Counterstain with DAPI to visualize nuclei

  • Quantify FOLR2+ macrophages in different tumor regions (tumor core vs. invasive margin)

• Functional Assays:

  • Sort FOLR2+ vs. FOLR2- macrophages from tumors to compare functional properties

  • Assess cytokine production profile

  • Evaluate tumor-promoting functions (angiogenesis, immunosuppression)

This approach has revealed important insights into how FOLR2-expressing macrophages contribute to tumor progression and may represent therapeutic targets.

What methodological considerations are important when using FOLR2 antibodies to monitor response to folate-targeted therapies?

When monitoring therapeutic responses involving FOLR2-targeted approaches:

• Baseline Assessment:

  • Quantify FOLR2 expression level and distribution in target tissues before treatment

  • Document subcellular localization (membrane vs. cytoplasmic) as this may affect drug accessibility

• Longitudinal Monitoring:

  • Use consistent antibody clones and detection protocols across timepoints

  • Consider both expression level changes and pattern alterations (e.g., internalization)

  • Include internal controls to normalize between samples/timepoints

• Resistance Mechanisms:

  • Monitor for altered glycosylation patterns that might affect antibody binding

  • Assess for emergence of FOLR2-negative populations using flow cytometry

  • Check for compensatory upregulation of other folate transporters

• Correlative Analyses:

  • Relate FOLR2 expression changes to clinical outcomes

  • Consider parallel assessment of downstream pathways (Akt/mTOR/S6K1) to understand molecular response mechanisms

These methodological approaches can provide mechanistic insights into how folate-targeted therapies affect FOLR2-expressing cells and identify resistance mechanisms.

What are the optimal protocols for using FOLR2 antibodies in multiplexed imaging applications?

Multiplexed imaging with FOLR2 antibodies requires careful optimization:

• Sequential Staining Approach:

  • Start with the lowest abundance target (often FOLR2) using higher antibody concentration

  • Use complete stripping or inactivation between rounds

  • Validate that each stripping step doesn't affect tissue morphology

• Simultaneous Staining Strategy:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • For example, use mouse anti-FOLR2 with rabbit anti-CD68 and goat anti-CD163

  • Select secondary antibodies with minimal cross-reactivity

  • Use spectral unmixing to resolve overlapping fluorophores

• Detection System Selection:

  • For chromogenic multiplexing: Use HRP-conjugated secondary antibodies with different chromogens

  • For fluorescent multiplexing: Select fluorophores with minimal spectral overlap

  • For mass cytometry: Consider metal-conjugated FOLR2 antibodies for highly multiplexed analysis

• Controls for Multiplexed Imaging:

  • Single-stain controls for each target to establish baseline signal

  • FMO (fluorescence minus one) controls to assess spectral overlap

  • Absorption controls when using multiple antibodies from the same species

These approaches enable visualization of FOLR2 in complex tissue microenvironments while preserving spatial relationships with other markers.

What factors affect the sensitivity and specificity of FOLR2 ELISA assays?

The performance of FOLR2 ELISA systems depends on several critical factors:

• Antibody Pair Selection:

  • Capture and detection antibodies should recognize distinct, non-overlapping epitopes

  • The biotin-labeled detection antibody dilution (typically 1:100) significantly affects sensitivity

  • Monoclonal antibody pairs often provide better specificity than polyclonal pairs

• Sample Preparation:

  • Fresh samples typically yield better results than frozen-thawed specimens

  • Proper sample dilution is critical - refer to linear dilution validation data showing 80-99% recovery rates across serum, EDTA plasma, and heparin plasma samples

  • Pre-clearing steps may be needed to reduce matrix effects

• Assay Optimization:

  • Incubation times (60 minutes for antibody binding, 30 minutes for SABC working solution)

  • Temperature control (37°C for critical steps)

  • Washing procedures (5 washes after SABC incubation is recommended)

• Detection System:

  • HRP-streptavidin conjugate working solution preparation timing is critical (prepare within 30 minutes of use)

  • TMB substrate quality and development time (typically until color develops)

  • Stopping reaction at consistent timepoints

When optimized, FOLR2 ELISA systems can achieve intra-assay CV<8% and inter-assay CV<10% .

How can researchers accurately quantify FOLR2 in complex biological samples using immunoassay techniques?

Accurate quantification of FOLR2 in complex samples requires:

• Standard Curve Optimization:

  • Use a 7-point standard curve with 2-fold serial dilutions

  • Include blank control (zero standard)

  • Ensure standard curve encompasses expected sample concentration range

  • Validate that curve follows expected four-parameter logistic fit

• Sample Matrix Considerations:

  • Different biological fluids may require specific optimization

  • Recovery rates: Serum (88-102%, avg 93%), EDTA plasma (88-99%, avg 94%), Heparin plasma (86-101%, avg 98%)

  • Consider parallel dilution curves to identify optimal dilution factors

• Interference Testing:

  • Evaluate potential interferents in your biological system

  • Test for high-dose hook effect with very high concentration samples

  • Consider heterophile antibody blockers for serum/plasma samples

• Data Analysis Approaches:

  • Use appropriate curve-fitting algorithms for standard curve

  • Account for dilution factors in final calculations

  • Consider spike-and-recovery experiments to validate accuracy

These considerations ensure reliable quantification of FOLR2 across diverse sample types and concentration ranges.

How can FOLR2 antibodies be used to investigate the role of folate receptors in cancer progression mechanisms?

FOLR2 antibodies enable sophisticated investigations into cancer biology:

• Expression Profiling Across Cancer Types:

  • Western blot and IHC analyses reveal differential FOLR2 expression patterns

  • Non-small cell lung cancer (NSCLC) cell lines show significantly increased FOLR2 expression compared to normal bronchial epithelial cells

  • Quantitative analysis can correlate expression levels with clinical outcomes

• Functional Studies:

  • siRNA knockdown approaches using FOLR2-specific siRNA demonstrate its functional role:

    • Reduced cell viability in NCI-H1650 NSCLC cells following FOLR2 silencing

    • Cell cycle alterations with decreased proliferation

• Signaling Pathway Analysis:

  • FOLR2 involvement in Akt/mTOR/S6K1 signaling pathways can be assessed using phospho-specific antibodies alongside FOLR2 detection

  • miRNA regulation studies show that miR-622 targets FOLR2 to regulate colorectal cancer cell cycle progression

• Therapeutic Target Validation:

  • Antibody-dependent cellular cytotoxicity assays against FOLR2-expressing cancer cells

  • Competitive binding assays with potential therapeutic compounds

  • In vivo xenograft models with FOLR2 manipulation show altered tumor growth

These approaches have revealed FOLR2 as both a potential biomarker and therapeutic target in multiple cancer types.

What methodological approaches enable investigation of FOLR2 in placental development and pregnancy-related disorders?

FOLR2 plays important roles in placental biology that can be studied using specialized techniques:

• Hofbauer Cell (Placental Macrophage) Analysis:

  • FOLR2 expression is decreased in placentas from pregnancies with severe preeclampsia

  • Dual immunofluorescence staining with macrophage markers (CD163, CD68) identifies FOLR2+ Hofbauer cells

  • Quantitative analysis correlates FOLR2+ cell density with clinical parameters

• Hormone Response Studies:

  • Glucocorticoids enhance CD163 expression in placental Hofbauer cells

  • Treatment-response studies require carefully optimized FOLR2 antibody protocols

• Toll-like Receptor Interactions:

  • Co-localization studies of FOLR2 with TLR proteins in placental macrophages

  • Functional assays measuring cytokine production by FOLR2+ placental macrophages following TLR stimulation

• Comparative Analysis in Normal vs. Pathological Pregnancy:

  • Western blot quantification of FOLR2 expression levels

  • Flow cytometric enumeration of FOLR2+ cell populations

  • Spatial distribution analysis using immunohistochemistry