FOLR1 Antibody, FITC conjugated

What is FOLR1 and why is it an important research target?

FOLR1 (Folate Receptor Alpha) is a glycosylated protein that mediates cellular uptake of folic acid and reduced folates. It represents a significant research target for several reasons:

• It is dramatically upregulated in many carcinomas, particularly ovarian cancer, making it valuable as a cancer biomarker

• It serves as a selective marker for midbrain dopamine (mesDA) neural progenitors during development

• The protein has a calculated molecular weight of 26.5 kDa but migrates as 35-43 kDa under reducing conditions due to glycosylation

• It exists in both membrane-bound and secreted forms, with a GPI anchor for membrane attachment

• FOLR1 has emerged as a therapeutic target for antibody-based cancer therapies

What are the key characteristics of FITC as a fluorescent conjugate for antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides researchers with specific optical properties that are advantageous for certain applications:

• Excitation wavelength: Approximately 488-499 nm

• Emission wavelength: Approximately 515-535 nm

• Compatible with the 488 nm spectral line of argon-ion lasers commonly used in flow cytometry

• Provides bright green fluorescence for visualization in fluorescence microscopy and flow cytometry applications

• The fluorochrome is sensitive to pH, with optimal fluorescence in slightly alkaline conditions

• FITC-labeled antibodies require protection from light and minimal freeze-thaw cycles to maintain optimal performance

What structural and functional properties characterize human FOLR1?

Human FOLR1 possesses several distinct structural and functional characteristics:

• Full protein length of 257 amino acids with a canonical mass of 29.8 kDa (unglycosylated)

• The functional region typically spans amino acids 25-234

• Contains multiple glycosylation sites that contribute to its apparent molecular weight of 35-43 kDa on SDS-PAGE

• Functions in folic acid binding and receptor activity

• Plays roles in vesicle-mediated transport and post-translational protein modification

• Tissue-specific expression observed in kidney, lung, placenta, and thymus

• Serves as a documented cancer marker with overexpression in various tumor types

What are the primary research applications for FITC-conjugated FOLR1 antibodies?

FITC-conjugated FOLR1 antibodies are versatile tools with multiple research applications:

• Flow cytometry for cell surface expression analysis and isolation of FOLR1-positive cell populations

• Immunocytochemistry (ICC) to visualize FOLR1 expression patterns in cultured cells

• Immunofluorescence (IF) for detection of FOLR1 in tissue sections

• Fluorescence-activated cell sorting (FACS) to isolate and enrich FOLR1-expressing cells

• Cancer biomarker research, particularly in ovarian, breast, and lung cancers

• Developmental biology studies, specifically for midbrain dopaminergic neurons identification

• Monitoring FOLR1 expression in response to therapeutic interventions

How can FITC-conjugated FOLR1 antibodies be used to identify and isolate mesDA neural progenitors?

FOLR1 antibodies have proven valuable for isolating midbrain dopamine (mesDA) neural progenitors:

• FolR1 is selectively expressed in mesDA progenitors both in vivo and in vitro

• In embryonic stem cell (ESC) differentiation models, FOLR1+ cells co-label with mesDA markers including Lmx1a, Dmrt5, and Pitx3

• FOLR1 does not co-stain with non-mesDA markers such as Nkx6.1, Islet1, Pax6, or Lim1/2

• Flow cytometry sorting of FOLR1+ cells yields populations highly enriched for Pitx3-GFP expressing cells

• Post-sorting analysis confirms that >80% of sorted cells express high levels of FOLR1

• Seven days post-sorting, FOLR1+ fractions maintain enrichment in Foxa2+Lmx1a+ cells, while FOLR1- fractions rarely contain these markers

• FOLR1+ fractions show enrichment in TH+ neurons, while FOLR1- fractions contain more GABAergic, serotonergic neurons, and astrocytes

What cell models are appropriate for studying FOLR1 expression and antibody validation?

Several cellular models have been validated for FOLR1 research:

• MCF-7 human breast cancer cell line shows consistent FOLR1 expression suitable for flow cytometry validation

• HeLa human cervical carcinoma cells express detectable levels of FOLR1 and are used for antibody validation

• Ovarian cancer cell lines are particularly valuable given the clinical relevance of FOLR1 in this cancer type

• Embryonic stem cell (ESC) differentiation models, especially those with reporter constructs like Lmx1a-GFP and Pitx3-GFP

• Anti-FOLR1 CAR-293 cells can be used to evaluate binding activity of FOLR1 antibodies and proteins

• Formalin-fixed, paraffin-embedded epithelial ovarian, fallopian tube, or primary peritoneal cancer tissue specimens for clinical studies

What is the optimal protocol for flow cytometry using FITC-conjugated FOLR1 antibodies?

For optimal flow cytometry results with FITC-conjugated FOLR1 antibodies:

• Cell preparation: Use 2-5 × 10^5 cells per sample in cold buffer containing PBS with 1-5% BSA or serum

• Controls: Include appropriate isotype controls (e.g., FITC-conjugated IgG of matching isotype) and unstained samples

• Staining: Incubate cells with 1-5 μg/mL of FITC-conjugated FOLR1 antibody for 30-60 minutes at 4°C in the dark

• Washing: Wash 2-3 times with buffer to remove unbound antibody

• Analysis: Use a flow cytometer with 488 nm laser excitation and detection in the 515-535 nm range

• Gating strategy: First gate on viable cells, then analyze FITC signal intensity compared to isotype control

• For sorting applications: Use proper controls for setting sorting gates, including cells from control differentiation and unstained mesDA differentiated culture

• Post-sort validation: Perform flow cytometry analysis on sorted cells to confirm enrichment of FOLR1+ population

How should FITC-conjugated FOLR1 antibodies be stored and handled to maintain optimal activity?

Proper storage and handling are critical for antibody performance:

• Storage temperature: Store at -20°C or below for long-term storage of lyophilized antibody

• For liquid formulations: Some products recommend 2-8°C storage without freezing

• Light protection: Always protect FITC-conjugated antibodies from light exposure

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles by preparing small aliquots before freezing

• Reconstitution: For lyophilized antibodies, reconstitute according to the Certificate of Analysis provided by the manufacturer

• Buffer considerations: Some formulations contain glycerol (50%) and may be stored at -20°C

• Working dilutions: Prepare fresh working dilutions on the day of the experiment

• Expiration: Adhere to the expiration date provided by the manufacturer

What quality control parameters should be evaluated when using FITC-conjugated FOLR1 antibodies?

Several quality control parameters should be assessed:

• Antibody purity: Should be >95% as determined by SDS-PAGE and/or SEC-MALS

• Binding specificity: Validate using positive control cells known to express FOLR1 (e.g., MCF-7, HeLa)

• Negative controls: Include appropriate isotype controls at the same concentration

• Signal-to-noise ratio: Evaluate the separation between positive and negative populations

• Lot-to-lot consistency: Test new lots against previous lots when possible

• FITC-to-protein (F/P) ratio: Optimal labeling without over-conjugation

• Functional activity: For example, testing the antibody's ability to bind immobilized Folic acid-BSA conjugate

• Species reactivity: Confirm that the antibody recognizes the appropriate species (human, mouse, etc.)

How can I distinguish between specific and non-specific binding when using FITC-conjugated FOLR1 antibodies?

To ensure accurate identification of specific binding:

• Always include matched isotype controls (e.g., FITC-conjugated IgG of the same isotype)

• Use cell lines known to be negative for FOLR1 expression as biological negative controls

• Perform titration experiments to determine the optimal antibody concentration that maximizes specific while minimizing non-specific binding

• Evaluate the staining pattern - FOLR1 should show membrane and cytoplasmic localization

• Consider blocking experiments with unconjugated anti-FOLR1 antibody or recombinant FOLR1 protein

• For FACS applications, include unstained controls and single-color controls for proper compensation

• Verify results using alternative detection methods (e.g., unconjugated primary antibody with secondary detection)

What factors may affect FOLR1 detection in flow cytometry experiments?

Several factors can influence FOLR1 detection and should be considered:

• FOLR1 expression level varies between cell types and can be affected by culture conditions

• Fixation methods may alter epitope accessibility - optimize fixation protocols for your specific antibody

• Cell cycle stage might influence FOLR1 expression levels

• In ESC-derived cultures, the developmental stage affects FOLR1 expression patterns (progressive increase in FolR1+ cells from day 8-14)

• After cell sorting, FOLR1 staining intensity may decrease over time (weaker at day 7 post-sort compared to day 1)

• Buffer composition and pH can affect FITC fluorescence intensity

• Antibody concentration must be optimized - too high can increase background, too low may miss low-expressing populations

• Spectral overlap with other fluorochromes in multi-color experiments requires proper compensation

How do I interpret varying FOLR1 expression levels in different experimental contexts?

Interpreting FOLR1 expression requires consideration of several factors:

• Expression patterns are cell type-specific - compare within similar cell types

• In developmental contexts, FOLR1 expression is dynamic and stage-dependent (e.g., in mesDA lineage)

• Not all cells in a positive population express FOLR1 uniformly - examine the distribution pattern rather than just mean values

• Quantify using mean/median fluorescence intensity (MFI) relative to isotype controls

• In mesDA progenitor studies, most but not all FOLR1+ cells are Lmx1a-GFP+, while not all Lmx1a-GFP+ cells are FOLR1+

• In therapeutic monitoring, both percentage of positive cells and intensity of expression may be relevant

• For FOLR1-targeted therapies, establish meaningful clinical cutoffs based on validated scoring methods

How does glycosylation affect FOLR1 antibody binding and detection?

Glycosylation has significant implications for FOLR1 detection:

• Human FOLR1 has a calculated MW of 26.5 kDa but migrates as 35-43 kDa under reducing conditions due to glycosylation

• Glycosylation patterns can differ between cell types and may affect epitope accessibility

• Some antibody clones may be sensitive to specific glycoforms of FOLR1

• Deglycosylation treatments prior to analysis may alter antibody binding properties

• Different detection methods (flow cytometry vs. Western blot) may have varying sensitivities to glycosylation state

• When selecting antibodies, consider whether they target glycosylation-dependent or independent epitopes

• Quality control by SEC-MALS can verify the glycosylation state of recombinant FOLR1 proteins used as standards

What are the considerations for multiplexing FITC-conjugated FOLR1 antibodies with other fluorescent markers?

When designing multiplex experiments:

• FITC emission overlaps with PE and other green fluorophores - careful compensation is required

• Consider using alternative conjugates for FOLR1 (APC , PE , or Alexa Fluor 488 ) depending on your panel design

• In reporter systems (e.g., Pitx3-GFP), FITC conjugates may not be optimal due to spectral overlap with GFP

• Use appropriate single-color controls for each fluorophore in your panel

• Sequential staining may be necessary if using multiple antibodies from the same species

• For complex panels, spectral cytometry or imaging cytometry may offer advantages over conventional flow cytometry

• Validate each antibody individually before combining in a multiplex panel

• Consider the relative expression levels of targets when designing panels to pair brightest fluorophores with lowest expressed antigens

How can FOLR1 antibodies contribute to therapeutic development research?

FOLR1 antibodies play crucial roles in therapeutic research:

• Target validation: Confirm FOLR1 expression in patient samples using immunohistochemistry with validated antibodies

• Patient stratification: FOLR1 antibodies help identify patients suitable for FOLR1-targeted therapies

• Development of therapeutic antibodies: Several FOLR1-targeting antibodies are in clinical development, including farletuzumab

• Mechanism studies: Understanding antibody-mediated effects like ADCC and CDC in FOLR1-positive tumors

• Monitoring therapy: Tracking changes in FOLR1 expression during treatment

• Resistance mechanisms: Identifying alterations in FOLR1 expression that may contribute to treatment resistance

• CAR-T cell development: FOLR1 antibodies are used in creating chimeric antigen receptors targeting FOLR1+ cancers

• Companion diagnostics: FOLR1 antibodies like VENTANA FOLR1 (FOLR1-2.1) RxDx Assay are used to determine eligibility for FOLR1-targeted therapies

What technical differences exist between various FITC-conjugated FOLR1 antibodies?

Important technical differences to consider when selecting an antibody:

• Clonality: Both monoclonal and polyclonal FITC-conjugated FOLR1 antibodies are available

• Immunogen: Most are raised against human FOLR1 protein (25-234 AA) , but epitopes may differ

• Host species: Commonly available from rabbit or mouse hosts

• Purification methods: Protein G purification is common for high-quality antibodies

• Species reactivity: Most are human-specific, but cross-reactivity varies between products

• Validated applications: Some are validated for specific techniques like flow cytometry, ICC, IF, or FACS

• Buffer formulations: May contain different preservatives (e.g., 0.03% Proclin 300) , stabilizers, or carrier proteins

• Storage recommendations: Some require -20°C storage, while others are stored at 2-8°C

• FITC conjugation efficiency may vary between products and affect brightness

Table 1: Comparison of Available FITC-Conjugated FOLR1 Antibody Products

*Note: This table represents products mentioned in the search results and is not exhaustive of all available products.

What are emerging research areas utilizing FITC-conjugated FOLR1 antibodies?

Several promising research directions are emerging:

• Single-cell analysis of FOLR1 expression heterogeneity in tumors and during development

• Spatial transcriptomics combined with FOLR1 immunofluorescence for comprehensive tissue analysis

• Development of improved FOLR1-targeted therapies including antibody-drug conjugates

• CRISPR-based studies of FOLR1 function in normal and disease states

• Applications in regenerative medicine using FOLR1 as a marker for isolating specific neural progenitors

• Multi-omics approaches integrating FOLR1 protein expression with genomic and transcriptomic data

• Advanced imaging techniques for in vivo monitoring of FOLR1-expressing cells

• Expansion of FOLR1-based diagnostics beyond ovarian cancer to other FOLR1-expressing malignancies

How can researchers optimize experiments combining FOLR1 antibodies with other research tools?

To maximize experimental value:

• Combine FOLR1 antibody staining with functional assays to correlate expression with cellular behaviors

• Integrate with genetic manipulation (CRISPR, RNAi) to study cause-effect relationships

• Pair with high-content imaging for morphological and subcellular localization analysis

• Use with patient-derived models to enhance translational relevance

• Combine flow cytometry sorting with downstream molecular analyses (RNA-seq, proteomics)

• Integrate with metabolic studies given FOLR1's role in folate uptake

• Correlate FOLR1 expression with drug sensitivity profiles in precision medicine approaches

• Utilize with computational biology for predictive modeling of FOLR1-related pathways