CSF2RB Recombinant Monoclonal Antibody

What is CSF2RB and why is it important in immunological research?

CSF2RB, also known as CD131, IL3RB, IL5RB, and the common beta chain, functions as a shared receptor subunit for multiple cytokines including GM-CSF, IL-3, and IL-5. This transmembrane glycoprotein forms heterodimeric complexes with cytokine-specific alpha chains to create functional high-affinity receptors that regulate critical processes in myeloid cell development, function, and survival.

The significance of CSF2RB in research stems from its central role in coordinating signals from multiple cytokines that regulate hematopoiesis, inflammation, and immune responses. Mutations in CSF2RB have been associated with pulmonary alveolar proteinosis (SMDP5), making it relevant for both basic immunology and disease-specific investigations. Understanding CSF2RB signaling provides insights into myeloid cell biology, inflammatory disorders, and potential therapeutic targets.

What are the key differences between recombinant monoclonal and polyclonal CSF2RB antibodies?

Recombinant monoclonal antibodies targeting CSF2RB offer distinct advantages over polyclonal alternatives, particularly for precise research applications:

What validated applications are supported by commercially available CSF2RB recombinant monoclonal antibodies?

Commercial CSF2RB recombinant monoclonal antibodies have been validated for multiple research applications, with specific optimization requirements for each technique:

For optimal results, protocol optimization is essential. For instance, in immunofluorescence applications, paraformaldehyde fixation (4%) followed by permeabilization with 0.1-0.3% Triton X-100 typically preserves CSF2RB epitopes while maintaining cellular architecture. Western blot applications may require optimization of reducing conditions depending on the epitope recognized by the specific antibody clone.

What host systems are used to produce CSF2RB recombinant monoclonal antibodies and why does this matter?

Various expression systems are employed to produce CSF2RB recombinant monoclonal antibodies, each offering distinct advantages:

The choice of expression system impacts antibody performance in specific applications. HEK293F-derived antibodies typically excel in applications requiring recognition of conformational epitopes due to proper mammalian folding and glycosylation . For applications like western blotting where denatured proteins are detected, E. coli-expressed antibodies may be sufficient. Baculovirus-expressed antibodies offer a middle ground with some post-translational modifications at potentially lower cost than mammalian systems .

What controls should be included when validating CSF2RB antibody specificity?

Rigorous validation of CSF2RB antibody specificity requires comprehensive controls to ensure experimental reliability:

For flow cytometry applications using conjugated antibodies (FITC, HRP, APC, or biotin-linked formats) , fluorescence-minus-one (FMO) controls are essential to establish proper gating strategies. In immunohistochemistry applications, tissue from CSF2RB knockout models provides the gold standard negative control, though this may not always be available.

Cross-reactivity testing with related proteins (IL3RA, IL5RA, CSF2RA) is particularly important given the structural similarities within this receptor family. This can be accomplished through western blotting or immunoprecipitation followed by mass spectrometry to confirm target identity.

How does epitope specificity of different CSF2RB antibody clones affect cytokine signaling studies?

The epitope specificity of CSF2RB antibodies significantly impacts cytokine signaling research outcomes through several mechanisms:

Research approaches requiring careful antibody selection include:

• Receptor internalization studies: Non-blocking antibodies that remain bound during endocytosis

• Heterodimerization analysis: Antibodies that don't interfere with alpha/beta chain interactions

• Phosphorylation detection: Compatible antibodies for simultaneous staining with phospho-specific antibodies

The search results indicate multiple CSF2RB antibody clones are available (including C1, C6, C8, C10, and C12) , likely recognizing different epitopes and thus suitable for different experimental purposes.

What are optimal protocol strategies for immunoprecipitation using CSF2RB recombinant monoclonal antibodies?

Successful immunoprecipitation (IP) of CSF2RB requires careful optimization of multiple parameters:

For transmembrane proteins like CSF2RB, lysis buffer composition is particularly critical. A starting formulation might include:

• 1% NP-40 or 0.5% Triton X-100 (mild enough to preserve protein-protein interactions)

• 150mM NaCl (physiological ionic strength)

• 50mM Tris-HCl pH 7.5

• Protease and phosphatase inhibitor cocktails

• 10% glycerol to stabilize proteins

For studying transient interactions or weakly associated complexes, chemical crosslinking prior to lysis can preserve interactions. This can be accomplished using membrane-permeable crosslinkers like DSP (dithiobis[succinimidyl propionate]) at 1-2mM for 30 minutes at room temperature before quenching and lysis.

Several CSF2RB monoclonal antibody clones have been validated for immunoprecipitation applications , enabling researchers to select optimal antibodies based on their specific experimental requirements.

How can CSF2RB antibodies be integrated into multiparameter flow cytometry panels?

Integrating CSF2RB detection into multiparameter flow cytometry requires strategic panel design considering spectral characteristics and antibody compatibility:

Panel development methodology should include:

• Titration optimization: Each conjugated antibody requires individual titration to determine optimal concentration. This involves testing serial dilutions (typically 5-fold) starting from manufacturer's recommendation and selecting the concentration that provides maximal separation between positive and negative populations with minimal background.

• Spillover compensation: Proper compensation controls are essential, particularly when using FITC-conjugated CSF2RB antibodies which have significant spillover into PE channels. Single-color controls using the same conjugate on the same cell type provide the most accurate compensation matrices.

• Co-expression analysis optimization: When studying CSF2RB alongside alpha chain partners (GM-CSFR alpha, IL-3R alpha, IL-5R alpha), select complementary fluorochromes with minimal spectral overlap. For example, APC-conjugated CSF2RB antibodies pair well with PE-conjugated alpha chain antibodies.

• Functional correlation panels: For signaling studies, compatibility with phospho-protein detection is essential. Methanol permeabilization required for some phospho-epitopes may affect certain CSF2RB epitopes, necessitating antibody clone selection specifically validated for compatibility with phospho-flow protocols.

The monoclonal nature of recombinant CSF2RB antibodies ensures consistent staining patterns between experiments, critical for longitudinal studies tracking receptor expression changes over time or treatment conditions.

What are the technical considerations for using CSF2RB antibodies in multiplexed imaging applications?

Multiplexed imaging with CSF2RB antibodies requires specialized techniques to overcome limitations of conventional fluorescence microscopy:

Methodological implementation considerations include:

• Antibody validation for tissue imaging: Recombinant monoclonal antibodies targeting CSF2RB must be validated specifically for tissue applications, as performance can differ significantly from flow cytometry. This includes optimization of:

  • Fixation protocols (4% PFA typically preserves CSF2RB epitopes)

  • Antigen retrieval methods (citrate vs. EDTA-based)

  • Blocking conditions to minimize background (typically 5-10% serum from secondary antibody species)

• Multiplexed receptor family analysis: Studying CSF2RB alongside alpha chains requires careful antibody selection to avoid cross-reactivity and ensure compatibility with multiplexing techniques. Options include:

  • Primary antibodies from different host species allowing simultaneous detection

  • Directly conjugated primary antibodies with spectrally distinct fluorophores

  • Sequential staining protocols with intermediate stripping or quenching steps

• Quantitative image analysis optimization:

  • Calibration standards for consistent quantification across experiments

  • Automated segmentation algorithms to identify membrane vs. cytoplasmic receptor localization

  • Colocalization analysis parameters for receptor complex studies

Several CSF2RB antibody clones have been specifically validated for immunofluorescence applications , providing researchers with options for different multiplexed imaging approaches.

How can CSF2RB antibodies be utilized to study receptor internalization and trafficking?

Studying CSF2RB internalization and trafficking requires specialized experimental approaches utilizing antibodies in dynamic cellular contexts:

Methodological implementation for internalization studies includes:

• Antibody feeding assay protocol:

  • Surface labeling: Incubate live cells with CSF2RB antibody at 4°C (prevents internalization)

  • Stimulation: Warm to 37°C with or without cytokine (GM-CSF, IL-3, IL-5) to trigger internalization

  • Differential detection: Apply differentially labeled secondary antibodies before and after permeabilization to distinguish surface from internalized receptors

  • Quantification: Flow cytometry or confocal microscopy for kinetic analysis

• Recycling assay optimization:

  • Initial labeling: Surface CSF2RB labeling at 4°C

  • Internalization: Temperature shift to permit endocytosis

  • Acid strip: Remove remaining surface antibody (pH 2.5 buffer)

  • Recycling phase: Return to 37°C to allow recycling

  • Detection: Appearance of labeled antibody at surface indicates recycling

• Endosomal colocalization analysis:

  • Antibody uptake: Allow labeled CSF2RB antibody to internalize

  • Fixation: Preserve spatial relationships (timing is critical)

  • Counter-staining: Label endosomal compartments (EEA1, Rab5, Rab7, LAMP1)

  • Analysis: Calculate Pearson's correlation coefficients for colocalization quantification

For these applications, selecting non-blocking antibody clones is crucial as blocking antibodies may artificially alter receptor trafficking patterns. The multiple monoclonal antibody clones available (C1, C6, C8, C10, C12) likely offer different properties in terms of internalization tracking capabilities.

What strategies exist for studying CSF2RB-associated signaling complexes using recombinant monoclonal antibodies?

Investigating CSF2RB-associated signaling complexes requires sophisticated approaches combining antibody-based techniques with other methodologies:

Implementing these approaches requires specific methodological considerations:

• Optimized co-immunoprecipitation protocol:

  • Gentle cell lysis (1% digitonin or 0.5% NP-40) to preserve complexes

  • Antibody selection targeting epitopes away from protein interaction domains

  • Optional crosslinking (1-2mM DSP) for stabilizing transient interactions

  • Sequential IPs to identify higher-order complexes

  • Controls with isotype-matched antibodies to identify non-specific binding

• Proximity ligation assay implementation:

  • Paired antibodies from different species (e.g., rabbit anti-CSF2RB with mouse anti-JAK2)

  • Carefully optimized fixation to preserve protein complexes while enabling antibody access

  • Quantification of PLA dots per cell as readout of interaction frequency

  • Correlation with activation status using phospho-specific antibodies

• Dynamic signaling complex analysis:

  • Time-course studies following cytokine stimulation

  • Membrane microdomain isolation (lipid rafts) to examine compartmentalized signaling

  • Inhibitor studies to determine complex dependency on specific interactions

The availability of multiple monoclonal antibody clones recognizing different CSF2RB epitopes enables researchers to select antibodies that don't interfere with complex formation while providing specific pulldown of receptor-associated proteins.

How can CSF2RB antibodies contribute to understanding and treating hematological disorders?

CSF2RB recombinant monoclonal antibodies serve as valuable tools for investigating hematological disorders with disrupted cytokine signaling:

Methodological implementation in disease research includes:

• Diagnostic application development:

  • Standardized flow cytometry panels incorporating CSF2RB detection alongside lineage markers

  • Quantitative immunohistochemistry protocols for pathology laboratories

  • Reference ranges for receptor expression across cell types and disease states

• Functional assessment protocols:

  • Ex vivo cytokine stimulation assays measuring STAT5 phosphorylation relative to receptor expression

  • Correlation of receptor function with clinical parameters and outcomes

  • Identification of patient subgroups based on receptor signaling profiles

• Therapeutic monitoring applications:

  • Tracking receptor expression/function during treatment with JAK inhibitors or other targeted therapies

  • Development of companion diagnostic assays for cytokine-based therapies

  • Assessment of receptor modulation as pharmacodynamic markers

Recombinant monoclonal antibodies provide the consistent performance essential for developing standardized clinical diagnostic protocols. While current CSF2RB antibodies are explicitly labeled "For Research Use Only" , the methodologies developed using these reagents can ultimately inform clinical test development.

The multiple available antibody formats, including unconjugated, FITC, HRP, APC, and biotin-linked versions , provide researchers flexibility to develop optimized protocols across different analytical platforms from flow cytometry to tissue imaging.