IL34 Human, His

What is the structure and function of human IL-34?

IL-34 is a secreted homodimeric glycoprotein that belongs to the short-chain helical hematopoietic cytokine family, though it shows no apparent consensus structural domains, motifs, or sequence homology with other cytokines . The protein has a calculated molecular weight of 27.3 kDa but migrates as 32 kDa and 35-40 kDa under reducing conditions due to glycosylation .

Functionally, IL-34 binds to three main receptors:

• CSF-1 receptor (CSF-1R)

• Receptor-type protein-tyrosine phosphatase-zeta (PTP-ζ)

• Chondroitin sulfate chains of syndecan-1

These interactions activate signaling pathways that regulate:

• Cellular proliferation, differentiation, and survival

• Metabolism and cytokine/chemokine expression

• Cellular adhesion and migration

In steady-state conditions, IL-34 contributes to the development and maintenance of Langerhans cells in the skin and microglia in the brain .

How does the His-tagged IL-34 differ from the native protein?

The His-tagged human IL-34 protein differs from the native form by containing a polyhistidine tag at the C-terminus . This modification allows for:

• Protein purification through immobilized metal affinity chromatography

• Detection of the protein using anti-His antibodies

• Immobilization of the protein onto surfaces for binding studies

Though the His tag is designed to minimally interfere with protein function, researchers should verify that the tag does not affect the biological activity of IL-34 in their specific application. Functional assays with the His-tagged IL-34 demonstrate that it retains binding activity to human M-CSF receptor with a linear range of 0.4-2 ng/mL and to human TREM2 with a linear range of 0.1-1 ng/mL .

What are the optimal storage conditions for human IL-34 protein with His tag?

For optimal stability of lyophilized His-tagged human IL-34:

• Store the lyophilized protein at -20°C or lower for long-term storage

• Avoid repeated freeze-thaw cycles that can damage protein structure and function

• Reconstitute according to the specific instructions provided in the Certificate of Analysis

• Typically reconstituted in PBS with 0.2 M Arginine (pH 7.4) with trehalose as a protectant

For working solutions:

• Store at 4°C for short-term use (1-2 weeks)

• Aliquot and store at -80°C for longer periods

• Validate stability in your specific buffer conditions if different from the recommended formulation

How can researchers effectively study IL-34 signaling pathways in human disease models?

Studying IL-34 signaling in human disease models requires a multi-faceted approach:

A. Expression analysis techniques:

• Quantitative PCR for mRNA expression

• ELISA for protein quantification (detection range: 100-2 pg/mL; lower limit of quantitation: 6 pg/mL for human serum samples with an initial dilution of 1:3)

• Immunohistochemistry for tissue localization

• Western blotting for protein expression and modification

B. Functional studies:

• Receptor binding assays using recombinant CSF-1R and PTP-ζ

• Phosphorylation assays to detect activation of downstream pathways

• Cell-based assays using monocyte differentiation models (IC50 for anti-IL34 = 30 ng/ml)

• Gene knockout or knockdown studies to assess functional relevance

C. Disease-specific considerations:

• For inflammatory bowel disease: assess IL-34 expression in intestinal tissue samples and correlate with disease severity

• For lung adenocarcinoma: evaluate IL-34 expression as a prognostic marker, as loss of expression correlates with poor prognosis

• For bone disorders: examine the impact on osteoclast and osteoblast differentiation as IL-34 plays a role in bone formation

What are the methodological challenges in distinguishing IL-34 functions from CSF-1 effects since they share the same receptor?

Differentiating IL-34 functions from CSF-1 effects presents several methodological challenges that researchers can address through the following approaches:

A. Selective inhibition strategies:

• Use specific blocking antibodies against IL-34 (e.g., phage-derived anti-mouse IL34 with 21.3 nM affinity) versus CSF-1 (e.g., rat anti-mouse CSF1 with Kd of 9.3 nM)

• Employ receptor mutants that selectively bind one ligand but not the other

• Design competitive binding assays to determine binding site overlap and differences

B. Tissue-specific expression analysis:

• Compare expression patterns of IL-34 versus CSF-1 across tissues

• Identify cellular sources unique to each cytokine

• Analyze temporal expression patterns during development or disease progression

C. Downstream signaling comparison:

• Perform phosphoproteomic analyses to identify unique signaling nodes

• Use CRISPR-based screens to identify differential genetic dependencies

• Conduct transcriptomic analyses after selective stimulation with each cytokine

D. Alternative receptor interactions:

• Study IL-34 binding to PTP-ζ, which is not shared with CSF-1

• Investigate the interaction with syndecan-1, another receptor specific to IL-34

• Design experimental systems that isolate these non-overlapping receptor interactions

How does IL-34 contribute to bone formation, and what experimental models best demonstrate this function?

IL-34 plays a significant role in bone formation, with several experimental approaches to study this function:

A. IL-34 knockout mouse models demonstrate:

• Severe growth delay and dysmorphoses in whole skeleton elements at 15 days old

• Specific alterations in craniofacial skeleton associated with hydrocephaly

• Significant reduction in skull growth in all planes (sagittal, vertical, and transversal)

• Reduction in long bone growth in both length and width dimensions

B. Histological analyses reveal:

• Increased TRAP staining (indicating osteoclastic cells)

• Increased Osterix/SP7 staining (indicating pre-osteoblastic cells)

• No difference in RUNX2-positive cells between knockout and wild-type mice, suggesting a slowdown of osteoblast differentiation

C. Blocking antibody studies show:

• Administration of IL-34 blocking antibody (Sheff.5 clone) during the first post-natal week induces skull growth alterations similar to those in knockout mice, though to a lesser extent

D. Recommended experimental models:

• Il34LacZ reporter mice for visualizing expression patterns

• Conditional knockout models for tissue-specific deletion

• In vitro osteoblast and osteoclast differentiation assays with IL-34 supplementation or inhibition

• MicroCT analysis for detailed bone structure assessment

What are the best practices for quantifying IL-34 in different biological samples?

Accurate IL-34 quantification in biological samples requires careful consideration of sample type and detection method:

For human serum samples:

• Use ELISA with anti-huIL34 (R&D Mab5265) as capture antibody

• Add heterophilic antibody blocker (Immunoglobulin Inhibiting Reagent) at 1 mg/ml to serum samples prior to dilution

• Detect with biotin-labeled hamster anti-IL34 followed by streptavidin-peroxidase

• Detection range: 100-2 pg/mL; LLOQ: 6 pg/mL (initial dilution 1:3)

• Note that up to 1 μg/ml of human soluble CSF1R does not interfere with detection

For mouse serum and tissue lysates:

• Use ELISA with sheep anti-muIL34 (R&D AF5195) as capture antibody

• Detect with biotin-labeled sheep anti-muIL34

• Detection range: 1,000-4 pg/mL; LLOQ: 12 pg/mL (initial dilution 1:3)

• Note that up to 1 μg/ml of murine soluble CSF1R and up to 1 mg/ml anti-IL34 do not interfere with detection

For cell culture supernatants:

• Culture cells (1 × 10^6) in a single well of a six-well plate in 2 ml of media

• Harvest media after 72 hours and clarify by centrifugation

• Use 50 μl of media for ELISA

For tissue samples:

• Process tissues consistently to minimize variation

• Consider using β-galactosidase staining for IL34 reporter mice (e.g., Il34LacZ)

• Use immunohistochemistry for spatial distribution analysis

How can researchers optimize binding studies between His-tagged IL-34 and its receptors?

Optimizing binding studies between His-tagged IL-34 and its receptors requires attention to several experimental parameters:

A. Surface plasmon resonance (SPR) considerations:

• Immobilize receptors (CSF-1R, PTP-ζ) on the sensor chip surface

• Use His-tagged IL-34 as the analyte in solution

• Start with a concentration range of 0.1-100 nM based on reported affinities

• Ensure proper regeneration conditions between binding cycles

• Control for non-specific binding using irrelevant proteins

B. ELISA-based binding assays:

• Immobilize Human M-CSF R, Mouse IgG2a Fc Tag at 5 μg/mL (100 μL/well)

• Titrate His-tagged IL-34 within the linear range of 0.4-2 ng/mL

• For TREM2 binding, immobilize Human TREM2, Fc Tag at 5 μg/mL

• Use a linear range of 0.1-1 ng/mL for TREM2 binding studies

C. Cell-based binding assays:

• Use flow cytometry with fluorescently labeled His-tagged IL-34

• Employ competition assays with unlabeled ligands to determine specificity

• Validate binding using receptor knockdown or knockout cells as controls

• Consider using cells that naturally express receptors versus transfected cells

D. Co-immunoprecipitation approaches:

• Use anti-His antibodies to pull down IL-34 complexes

• Probe for receptor presence using specific antibodies

• Include appropriate controls (e.g., lysates without His-tagged proteins)

• Consider crosslinking for transient interactions

What experimental approaches can resolve contradictory findings in IL-34 research across different disease models?

Resolving contradictory findings in IL-34 research requires systematic approaches:

A. Standardization of experimental methods:

• Use consistent cell lines, primary cells, or animal models

• Standardize protein preparations (His-tagged versus untagged)

• Employ consistent protocols for cytokine stimulation (concentration, duration)

• Utilize identical readout systems across laboratories

B. Context-dependent analysis:

• Evaluate IL-34 effects in different tissue microenvironments

• Consider the influence of inflammatory status on IL-34 signaling

• Account for species-specific differences (human versus mouse)

• Assess the impact of disease stage on IL-34 function

C. Comprehensive receptor analysis:

• Determine the relative expression levels of CSF-1R versus PTP-ζ in different models

• Examine receptor signaling components in various cell types

• Consider the role of receptor internalization and turnover

• Evaluate the impact of soluble receptor forms

D. Integrated multi-omics approach:

• Combine transcriptomics, proteomics, and metabolomics data

• Use systems biology tools to identify context-specific signaling networks

• Employ machine learning algorithms to identify patterns in complex datasets

• Validate key findings using orthogonal experimental approaches