CSF2RA Antibody

What is CSF2RA and what are its recognized alternative designations in scientific literature?

CSF2RA (Colony Stimulating Factor 2 Receptor Alpha) is the low-affinity receptor component for granulocyte-macrophage colony-stimulating factor (GM-CSF). It has multiple synonyms in scientific literature including: CD116, CDw116, CSF2R-A, GM-CSFR, CSF2RAX, CSF2RAY, CSF2RX, CSF2RY, GMCSFR, and GMR . The receptor transduces signals that result in the proliferation, differentiation, and functional activation of hematopoietic cells . The human CSF2RA gene has been assigned Gene ID 1438 and corresponds to UniProt accession number P15509 .

Which cell types predominantly express CSF2RA and at what molecular weight is it typically detected?

CSF2RA is primarily expressed on neutrophils, eosinophils, monocytes, and macrophages, making it an important marker for myeloid lineage cells . While the calculated molecular weight of CSF2RA is approximately 47 kDa, the observed molecular weight in experimental conditions is typically higher—55 kDa according to Proteintech's antibody validation data and approximately 80 kDa as reported by antibodies-online . This discrepancy likely reflects post-translational modifications, particularly glycosylation, which are common for cell surface receptors.

What are the validated applications for commercially available CSF2RA antibodies?

Available CSF2RA antibodies have been validated for multiple experimental applications as summarized in the following table:

WB: Western Blot; IHC: Immunohistochemistry; ICC: Immunocytochemistry; IP: Immunoprecipitation; CyTOF: Cytometry by Time of Flight

What are the recommended dilutions and protocols for CSF2RA antibody applications?

Optimal dilutions vary by application and specific antibody. For Proteintech's 18307-1-AP antibody:

These dilutions should be considered starting points. Researchers should perform titration experiments to determine optimal concentrations for their specific experimental conditions . For IHC applications with this antibody, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used as an alternative .

How should researchers troubleshoot inconsistent CSF2RA detection in Western blot experiments?

When experiencing difficulty detecting CSF2RA in Western blot experiments, consider these methodological adjustments:

• Sample preparation: CSF2RA is a membrane protein; ensure adequate cell lysis and protein solubilization with appropriate detergents.

• Sample source verification: Validate experimental samples using positive controls such as HL-60 or THP-1 cells, which have been confirmed to express detectable levels of CSF2RA .

• Antigen integrity: CSF2RA may be sensitive to repeated freeze-thaw cycles; use freshly prepared samples when possible.

• Buffer optimization: For storage and experimental preparation, use buffer systems that maintain protein stability, such as PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Blocking optimization: If background is high, adjust blocking conditions or consider alternative blocking agents to improve signal-to-noise ratio.

How can CSF2RA antibodies be applied in studying pulmonary alveolar proteinosis pathophysiology?

CSF2RA antibodies serve as crucial tools for investigating hereditary pulmonary alveolar proteinosis (hPAP), a rare lung disease caused by mutations in the CSF2RA gene. These antibodies can:

• Validate gene correction approaches: Confirm restoration of CSF2RA expression following lentiviral-mediated gene transfer in experimental models .

• Monitor therapy efficacy: Assess CSF2RA expression in pulmonary macrophage transplantation (PMT) studies, where gene-corrected macrophages are used as therapy for CSF2RA deficiency .

• Evaluate protein localization: Determine whether transplanted macrophages properly express and localize CSF2RA after engraftment in the lungs of Csf2ra knockout mice .

• Assess immune responses: Detect potential anti-transgene product antibodies that might develop following gene therapy approaches .

Research by Suzuki et al. demonstrated that lentiviral-mediated Csf2ra cDNA transfer successfully restored GM-CSF signaling in macrophages, with transplanted cells remaining within the lungs and not undergoing uncontrolled proliferation—validating this approach as a potential therapy for hPAP .

What experimental design considerations are important when studying CSF2RA in different disease models?

When designing experiments to investigate CSF2RA in disease models, researchers should consider:

• Selection of appropriate controls: Include both wild-type (WT) and knockout/disease models to properly assess interventions. The toxicology study by Suzuki et al. utilized both Csf2ra KO and WT mice groups for comprehensive evaluation .

• Dose determination: Establish dose-response relationships through single ascending dose (SAD) and repeat ascending dose (RAD) studies. In the published model, researchers evaluated 500,000 (target dose) and 5,000,000 (safety margin dose) cells per mouse .

• Temporal assessment: Design experiments with both short-term (acute) and long-term (chronic) evaluation timepoints. The referenced study included 24-hour, 14-day, and 6-month assessment periods to capture both immediate and delayed effects .

• Multifaceted outcome measures: Incorporate measurements of:

  • Functional parameters (e.g., pulmonary function)

  • Cellular parameters (e.g., inflammation markers)

  • Molecular parameters (e.g., transgene expression)

  • Safety parameters (e.g., adverse events)

• Sex-based differences: Account for potential sex differences in response; the referenced study noted behavioral differences between male and female mice that affected adverse event rates .

How do post-translational modifications affect CSF2RA detection and function?

Post-translational modifications significantly impact both the detection and functional properties of CSF2RA:

• Detection implications: The discrepancy between calculated (47 kDa) and observed molecular weights (55-80 kDa) primarily results from glycosylation . Researchers should anticipate band migration differences based on glycosylation state and experimental conditions.

• Epitope accessibility: Glycosylation may mask antibody binding sites, particularly for antibodies targeting extracellular domains. The monoclonal antibody 4H1 specifically recognizes an extracellular epitope of human CD116 , which may be affected by glycosylation status.

• Functional consequences: Glycosylation affects receptor trafficking, ligand binding affinity, and downstream signaling. When studying receptor function, researchers should consider native glycosylation patterns in their experimental systems.

• Experimental approaches: To assess the impact of glycosylation:

  • Compare migration patterns before and after treatment with glycosidases

  • Use cell lines with different glycosylation capacities

  • Compare recombinant proteins expressed in different host systems

What key validation criteria should researchers apply when selecting CSF2RA antibodies?

When selecting a CSF2RA antibody for research applications, evaluate these critical validation parameters:

• Application-specific validation: Confirm the antibody has been validated for your specific application (WB, IHC, FACS, etc.) with demonstrated results .

• Species reactivity: Verify reactivity with your experimental species. Many CSF2RA antibodies are human-specific with limited cross-reactivity to other species .

• Epitope information: Consider the epitope location—antibodies recognizing different domains may perform differently depending on experimental conditions. For example, Biomatik's antibody targets Arg28~Pro112 , while other antibodies may target different regions.

• Clonality appropriateness: Select monoclonal antibodies for highly specific detection of single epitopes, or polyclonal antibodies for detecting denatured proteins or capturing broader epitope ranges .

• Positive control validation: Confirm the antibody detects CSF2RA in established positive controls such as:

  • Cell lines: HL-60 cells, THP-1 cells

  • Tissues: Human placenta tissue

• Purity specifications: Higher purity antibodies typically provide more consistent results. The antibody from antibodies-online reports >95% purity by SDS-PAGE .

How can researchers distinguish between CSF2RA detection and non-specific binding?

To differentiate specific CSF2RA detection from non-specific binding:

• Include multiple controls:

  • Positive controls (cells/tissues known to express CSF2RA)

  • Negative controls (CSF2RA knockout or cells without expression)

  • Isotype controls (particularly for flow cytometry applications)

  • Blocking peptide controls (pre-incubation with immunizing peptide)

• Validate with orthogonal methods: Confirm protein expression using multiple techniques (e.g., Western blot, IHC, and flow cytometry) or with antibodies recognizing different epitopes.

• Optimize experimental conditions:

  • For Western blot: Adjust blocking conditions, antibody concentrations, and wash stringency

  • For IHC: Test different antigen retrieval methods (e.g., TE buffer pH 9.0 versus citrate buffer pH 6.0)

  • For flow cytometry: Carefully set gates using appropriate controls

• Genetic validation: When possible, use gene knockdown/knockout systems or cells from patients with CSF2RA mutations to confirm antibody specificity.

How do CSF2RA gene mutations contribute to hereditary pulmonary alveolar proteinosis (hPAP), and what experimental models exist to study this?

CSF2RA mutations disrupt GM-CSF signaling in alveolar macrophages, impairing surfactant clearance and leading to surfactant accumulation in the lungs (hPAP). The primary experimental model is the Csf2ra knockout (KO) mouse, which develops a phenotype resembling human hPAP .

Key experimental approaches to study this pathophysiology include:

• Gene correction studies: Lentiviral vector-mediated Csf2ra cDNA transfer has been shown to restore GM-CSF signaling in macrophages derived from Csf2ra KO mice .

• Cell therapy models: Pulmonary macrophage transplantation (PMT) studies have demonstrated that transplanted gene-corrected macrophages can engraft in the lungs of Csf2ra KO mice, replace dysfunctional endogenous macrophages, and correct the hPAP phenotype .

• Toxicology assessment frameworks: Researchers have established protocols for evaluating safety through single ascending dose (SAD) and repeat ascending dose (RAD) studies, with short-term (24h, 14d) and long-term (6m) evaluation timepoints .

• Pharmacokinetic/pharmacodynamic evaluations: Experiments tracking cell engraftment, biodistribution, and therapeutic efficacy can inform therapeutic potential and mechanism of action .

Recent studies have established a no observed adverse effect level (NOAEL) and a 10-fold safety margin for PMT of Csf2ra gene-corrected macrophages, supporting the development of this approach as a potential therapy for human hPAP .

What are the critical considerations when using CSF2RA antibodies in multiparametric flow cytometry experiments?

When incorporating CSF2RA antibodies into multiparametric flow cytometry panels:

• Fluorophore selection: Choose fluorophores with minimal spectral overlap with other markers in your panel. Consider brightness hierarchy—place brighter fluorophores on less abundantly expressed targets.

• Clone compatibility: Verify that the selected clone (e.g., 4H1) doesn't interfere with other antibodies in the panel through steric hindrance.

• Titration optimization: Perform antibody titration experiments to determine the optimal concentration that maximizes signal-to-noise ratio.

• Compensation controls: Prepare single-stained controls for each fluorophore using the same cells as your experimental samples.

• FMO controls: Include fluorescence minus one (FMO) controls to accurately set gates, particularly important for CSF2RA which may show variable expression levels.

• Fixation sensitivity: Test whether the epitope recognized by your CSF2RA antibody is sensitive to fixation procedures, as this may affect detection in protocols requiring fixation.

• Panel design considerations: Position CSF2RA strategically in your gating hierarchy based on whether it's being used as a lineage marker or for quantitative expression analysis.

• Data analysis approach: For quantitative analysis, consider reporting data as molecules of equivalent soluble fluorochrome (MESF) rather than arbitrary units to facilitate cross-experimental comparison.

What strategies can researchers employ when CSF2RA antibodies show inconsistent or unexpected results across different experimental platforms?

When facing inconsistent results with CSF2RA antibodies across platforms:

• Antibody validation reassessment:

  • Verify the antibody has been validated for each specific application

  • Consider using multiple antibodies targeting different epitopes

  • Test the antibody in well-characterized positive control samples

• Protocol optimization by application:

  • For Western blot: Compare reducing vs. non-reducing conditions, adjust protein loading amounts

  • For IHC: Evaluate both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

  • For flow cytometry: Optimize fixation/permeabilization methods if necessary

• Sample preparation considerations:

  • Membrane proteins like CSF2RA require efficient extraction procedures

  • Compare fresh vs. frozen samples to assess preservation effects

  • Evaluate different lysis buffers for optimal protein extraction

• Experimental variables documentation:

  • Record lot numbers of antibodies and reagents

  • Document incubation times and temperatures

  • Note sample handling procedures and storage conditions

• System-specific adaptations:

  • For cell lines: Verify expression levels before experiments

  • For primary cells: Account for donor variability and isolation methods

  • For tissues: Consider fixation effects on epitope accessibility

How should researchers interpret discrepancies in molecular weight observations for CSF2RA across different studies?

When encountering molecular weight discrepancies for CSF2RA:

• Expected variation range: Understand that CSF2RA's theoretical molecular weight (47 kDa) differs from observed weights in experimental settings (55-80 kDa) due to:

  • Post-translational modifications, particularly glycosylation

  • Different experimental conditions affecting protein migration

  • Tissue-specific or cell-specific variations in processing

• Technical factors to consider:

  • Gel percentage affects migration patterns

  • Running buffer composition influences mobility

  • Sample preparation methods (heat, reducing agents) impact structure

• Biological interpretations:

  • Different isoforms may be expressed in different tissues

  • Activation state may alter post-translational modification patterns

  • Disease states can affect protein processing

• Verification approaches:

  • Enzymatic deglycosylation to determine contribution of glycans

  • Mass spectrometry to confirm protein identity and modifications

  • Comparison with recombinant proteins of known molecular weight

• Reporting best practices:

  • Always report both observed and expected molecular weights

  • Document experimental conditions that may affect migration

  • Consider providing images of full blots with molecular weight markers