C1QTNF9B Antibody

What are the optimal applications for C1QTNF9B antibodies in experimental settings?

C1QTNF9B antibodies have demonstrated efficacy in several experimental applications with varying success rates:

• Western Blotting (WB): Most commercially available C1QTNF9B antibodies are validated for WB applications, with recommended dilution ranges of 1:500-2000 . This technique effectively detects the protein at approximately 35-42 kDa .

• ELISA: Many C1QTNF9B antibodies perform well in ELISA with recommended dilution ranges of 1:5000-20000 .

• Immunofluorescence (IF): Some antibodies are validated for immunofluorescence applications, including both cell staining (IF/ICC) and tissue sections .

• Immunohistochemistry (IHC): Select antibodies have been validated for paraffin-embedded (IHC-p) and frozen sections (IHC-f) .

When selecting an application, consider the protein's cellular localization. C1QTNF9B primarily localizes to secretory pathways and forms heteromeric complexes with CTRP9A or adiponectin required for secretion. Otherwise, it remains in the endoplasmic reticulum .

How should researchers optimize sample preparation for C1QTNF9B detection?

Optimal sample preparation varies by application and source material:

For Western Blot Analysis:

• Use standard lysis buffers containing protease inhibitors

• For serum samples, samples should be prepared under reducing conditions using immunoblot buffer groups like Buffer Group 1

• PVDF membranes have shown better results than nitrocellulose for C1QTNF9B detection

• When probing, use antibody concentrations of approximately 2 μg/mL for primary antibodies

For Tissue Samples:

• For immunohistochemistry, 7 μm sections show optimal results

• Fixation with formalin followed by blocking with 3% BSA solution improves specificity

• Overnight incubation with primary antibody at 4°C followed by 2-hour incubation with secondary antibody at room temperature

Serum/Plasma Samples:

• For analyzing circulating C1QTNF9B, collect samples after 6-hour fasting for standardized results

• C1QTNF9B can form heterotrimers with adiponectin, which may require specialized immunoprecipitation approaches to distinguish complexes

What validation methods confirm C1QTNF9B antibody specificity?

Comprehensive validation includes:

• Western blot verification: A specific band should be detected at approximately 35-42 kDa. Studies demonstrate successful detection in human serum samples showing bands at approximately 42 kDa .

• Positive controls: Use tissues with known expression (adipose tissue, cardiac endothelial cells) as positive controls .

• Knockout validation: Compare staining patterns between wild-type and C1QTNF9B knockout mice tissues to confirm specificity .

• Cross-reactivity assessment: Test antibodies against related CTRP family members, particularly C1QTNF9A, which shares high sequence homology.

• Peptide competition: Pre-incubation with immunizing peptide should abolish signal in applications like WB or IHC.

• Immunoprecipitation followed by mass spectrometry: For definitive validation of antibody specificity.

What are the critical storage considerations for C1QTNF9B antibodies?

Proper storage significantly impacts antibody performance and shelf-life:

Additional recommendations:

• Use manual defrost freezers rather than auto-defrost to prevent temperature fluctuations

• Aliquot reconstituted antibodies to minimize freeze-thaw cycles

• Store in glycerol-containing buffers (many commercial preparations contain 50% glycerol)

• Some formulations contain PBS with 0.02% sodium azide as preservative

What are the recommended dilution ranges for different C1QTNF9B antibody applications?

Optimal dilutions vary by application and specific antibody:

Remember that optimal dilutions should always be determined experimentally for each specific antibody, application, and laboratory condition .

What methodological approaches help distinguish between C1QTNF9A and C1QTNF9B in experimental settings?

Differentiating between these highly similar paralogs requires specialized techniques:

• Selective antibody targeting: Use antibodies raised against unique epitopes in the variable N-terminal regions where sequence divergence is greatest .

• PCR-based discrimination: Design primers targeting non-homologous regions:

  • C1QTNF9A-specific forward: 5'-CTGAGGAGTGGAACTTCGAG-3'

  • C1QTNF9B-specific forward: 5'-GAGTACAAGCGCATCTTCGAG-3'

  • Common reverse: 5'-CTGGTACACCTTGTAGCCGTG-3'

• Knockout models: Utilize C1QTNF9B knockout mice as negative controls while maintaining C1QTNF9A expression .

• Functional discrimination:

  • C1QTNF9B requires heteromeric complex formation for secretion

  • C1QTNF9A can be secreted independently

  • Co-immunoprecipitation studies can help identify specific binding partners

• Proteomic analysis: Mass spectrometry can identify peptide fragments unique to each paralog after tryptic digestion.

These approaches should be used in combination for reliable discrimination between these closely related proteins.

How can researchers effectively study C1QTNF9B's role in diabetic cardiomyopathy?

Investigation of C1QTNF9B in diabetic cardiomyopathy requires multi-faceted experimental designs:

• Animal models: High-fat diet (HFD) models effectively induce early stages of diabetic cardiomyopathy. Use 60 kcal-% fat diet (#D12492) for 12 weeks starting with 7-9 week-old mice .

• Genetic manipulation approaches:

  • CTRP9 knockout mice exhibit exacerbated insulin resistance and diastolic dysfunction

  • AAV9-mediated cardiac-specific overexpression (1×10^12 vg administered intravenously) ameliorates cardiomyopathy

• Functional assessment techniques:

  • Echocardiography for diastolic dysfunction evaluation

  • 18F-fluoro-deoxyglucose (FDG) positron emission tomography (PET) for glucose uptake measurement

  • Seahorse metabolic analyzer for mitochondrial function assessment

• Signaling pathway analysis:

  • Western blotting for phosphorylation status of AKT (T308, S473), S6 (S240/244, S235/236), GSK, and ERK1/2

  • Normalize to total protein levels and GAPDH as loading control

• Inflammation assessment:

  • RNA sequencing to identify inflammatory gene signatures

  • Immunofluorescence with CD45 antibodies (1:50 dilution) to quantify leukocyte infiltration

This comprehensive approach helps elucidate C1QTNF9B's protective effects against diabetic cardiomyopathy.

What experimental designs best assess C1QTNF9B-mediated AMPK activation?

AMPK activation by C1QTNF9B can be rigorously evaluated using:

• Cell-based assays:

  • Treat cardiomyocytes, skeletal muscle cells, or adipocytes with recombinant C1QTNF9B (recommended dose range: 0.1-10 μg/mL)

  • Assess time-course (5, 15, 30, 60 min) and dose-response relationships

• Phosphorylation analysis:

  • Western blotting for phospho-AMPK (Thr172) and downstream targets like ACC (Ser79)

  • Normalize to total AMPK and ACC protein levels

• Functional readouts:

  • Glucose uptake assays using labeled 2-deoxyglucose

  • Fatty acid oxidation measurements

  • Mitochondrial respiration using Seahorse analyzer with UK5099 (2 μM) as inhibitor control

• Specificity controls:

  • AMPK inhibitor Compound C (10 μM) should abolish effects

  • AMPK-null cells as negative controls

  • Comparison with known AMPK activators (AICAR, metformin) as positive controls

• In vivo validation:

  • Administer recombinant C1QTNF9B to mice (0.5 μg/g body weight, i.p.)

  • Tissue collection after 30-60 minutes for phospho-AMPK assessment

  • Compare with AMPK activators (AICAR, 0.5 mg/g body weight)

These approaches provide robust assessment of C1QTNF9B's AMPK-activating properties across multiple experimental systems.

How should researchers design experiments to investigate C1QTNF9B's paracrine effects in cardiac tissue?

Investigating paracrine signaling requires sophisticated experimental designs:

• Cell-specific expression analysis:

  • Single-cell RNA sequencing to identify cell types expressing C1QTNF9B

  • Immunofluorescence co-localization with cell-type markers (endothelial cells: CD31; cardiomyocytes: troponin T)

• Conditioned media experiments:

  • Culture cardiac endothelial cells (primary expressers of C1QTNF9B)

  • Transfer conditioned media to cardiomyocytes

  • Assess signaling pathway activation (AKT, AMPK, ERK1/2)

  • Compare with C1QTNF9B-depleted media (immunodepletion) as control

• Co-culture systems:

  • Transwell co-culture of endothelial cells with cardiomyocytes

  • Use C1QTNF9B knockout endothelial cells as negative control

  • Analyze cardiomyocyte function (calcium handling, contractility)

• In vivo approaches:

  • Cell-specific conditional knockout models (endothelial-specific Cre)

  • AAV9-mediated cardiac-specific overexpression

  • Assess cardiomyocyte responses through phospho-protein analysis and functional assays

• Receptor identification:

  • AdipoR1 is a likely receptor—use AdipoR1 knockdown/knockout to verify

  • Perform binding studies with labeled C1QTNF9B

  • Investigate receptor clustering and downstream signaling complex formation

These approaches collectively elucidate the paracrine mechanisms by which endothelial-derived C1QTNF9B influences cardiomyocyte function.

What considerations apply when analyzing C1QTNF9B in multi-protein complexes?

C1QTNF9B forms complex structures requiring specialized analytical approaches:

• Native protein complex preservation:

  • Use non-denaturing lysis buffers

  • Avoid reducing agents when studying homotrimers or heterotrimers

  • Consider native PAGE for intact complex separation

• Co-immunoprecipitation strategies:

  • Use antibodies against C1QTNF9B for pull-down

  • Alternatively, target known partners (CTRP9A, adiponectin)

  • Elute under mild conditions to maintain complex integrity

• Size exclusion chromatography:

  • Separate complexes by molecular weight (homotrimers ~120 kDa, higher-order multimers >200 kDa)

  • Collect fractions for further analysis

• Crosslinking approaches:

  • Use chemical crosslinkers (BS3, DSS) to stabilize transient interactions

  • Optimize crosslinker concentration and reaction time

• Advanced analytical techniques:

  • Blue native PAGE for separation of intact complexes

  • Mass spectrometry techniques:

    • Intact mass measurement for complex stoichiometry

    • Crosslink-MS to map interaction interfaces

    • Native MS for complex composition

• Functional analysis of complexes:

  • Compare signaling properties of homotrimers vs. heterotrimers with adiponectin

  • Investigate receptor binding preferences of different complex forms

These methodologies help elucidate C1QTNF9B's complex biology in its natural higher-order structures.

What gene knockout strategies are most effective for studying C1QTNF9B function?

Several genetic approaches offer insights into C1QTNF9B biology:

• Conventional knockout mouse models:

  • Complete gene deletion strategies have successfully been used to study C1QTNF9B

  • Phenotyping should include metabolic parameters (glucose tolerance, insulin sensitivity) and cardiac function

• Conditional knockout approaches:

  • Cell-specific deletion using Cre-loxP system

  • Endothelial-specific knockout using Tie2-Cre or Cdh5-CreERT2

  • Adipocyte-specific using Adipoq-Cre

  • Temporal control with tamoxifen-inducible CreERT2 systems

• AAV-mediated overexpression:

  • AAV9 vectors achieve cardiac-specific expression

  • Recommended dose: 1×10^12 viral genomes administered intravenously

  • Use TnT (troponin T) promoter for cardiomyocyte-specific expression

• CRISPR/Cas9 genome editing:

  • For generating cell line models

  • Design guide RNAs targeting early exons

  • Verify knockout by sequencing and protein expression analysis

• Knockin reporter strategies:

  • GFP or luciferase reporters to monitor expression patterns

  • Epitope tags (HA, FLAG) for tracking protein localization and interaction studies

Each approach offers distinct advantages for investigating different aspects of C1QTNF9B biology, from tissue-specific roles to temporal expression patterns.

How can researchers effectively measure C1QTNF9B expression changes across different disease models?

Comprehensive expression analysis requires multiple complementary approaches:

• Quantitative PCR protocols:

  • Recommended primer sequences:

    • Forward: 5'-ACGCCTGGCTCTGTACTTCT-3'

    • Reverse: 5'-TCTCCAGGGTGAACATCTCG-3'

  • Reference genes: GAPDH, β-actin, 18S rRNA

  • Analyze using 2^-ΔΔCt method

• Protein quantification methods:

  • Western blotting with serial dilutions of recombinant standards

  • ELISA with appropriate antibody pairs

    • Capture antibody: polyclonal anti-C1QTNF9B

    • Detection antibody: biotin-conjugated anti-C1QTNF9B

• Tissue expression analysis:

  • Immunohistochemistry with standardized protocols

  • Single-cell RNA sequencing for cell-type-specific expression

  • In situ hybridization for spatial resolution

• Disease model considerations:

  • Diabetic models: HFD feeding (12 weeks), STZ-induced diabetes, db/db mice

  • Cardiac injury models: TAC, MI, I/R injury

  • Standardize collection timepoints (e.g., after 6-hour fasting)

• Circulating levels assessment:

  • Collect serum under standardized conditions (time of day, fasting status)

  • Use sandwich ELISA with validated antibody pairs

  • Account for complex formation with adiponectin

These methodologies provide comprehensive insight into C1QTNF9B regulation across physiological and pathological states.

What challenges exist in developing function-blocking antibodies against C1QTNF9B?

Developing effective function-blocking antibodies presents several technical challenges:

• Epitope selection considerations:

  • Target functional domains (C1q globular domain) responsible for receptor binding

  • Avoid collagenous domains that may cross-react with other CTRP family members

  • Consider accessibility in native protein conformations

• Validation strategies:

  • Receptor binding inhibition assays

  • Signaling pathway inhibition (pAMPK, pAKT reduction)

  • Functional assays (glucose uptake, fatty acid oxidation)

  • Comparison with genetic knockout models

• Technical limitations to address:

  • Accessibility issues due to complex formation

  • Cross-reactivity with C1QTNF9A and other family members

  • Potential for compensatory upregulation of related proteins

  • Need for appropriate controls (isotype-matched, pre-immune serum)

• Administration considerations for in vivo studies:

  • Optimal antibody dose and administration schedule

  • Tissue penetration challenges, especially for cardiac studies

  • Half-life determination for dosing intervals

  • Verification of target engagement in tissues of interest

Researchers should employ multiple complementary approaches to confirm antibody-mediated functional blockade, including both in vitro and in vivo validation methods.

How can proteomic approaches enhance C1QTNF9B research?

Advanced proteomic techniques offer powerful insights into C1QTNF9B biology:

• Post-translational modification mapping:

  • C1QTNF9B contains multiple hydroxylated proline residues in its collagenous region

  • Mass spectrometry identification of:

    • Hydroxylation sites

    • Glycosylation patterns

    • Potential phosphorylation sites

• Interactome analysis:

  • Proximity labeling approaches (BioID, APEX)

  • Immunoprecipitation followed by mass spectrometry

  • Crosslinking mass spectrometry for interaction interfaces

  • Comparative analysis between normal and disease states

• Structural proteomics:

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Native mass spectrometry for complex stoichiometry

  • Ion mobility for structural characterization of assemblies

• Secretome analysis:

  • Quantitative proteomics of conditioned media

  • SILAC labeling for accurate quantification

  • Comparison between wild-type and C1QTNF9B knockout cells

• Clinical proteomics applications:

  • Multiple reaction monitoring (MRM) assays for absolute quantification

  • Parallel reaction monitoring (PRM) for increased specificity

  • Development of clinical assays for biomarker applications

These proteomic approaches provide molecular-level insights that complement traditional biochemical and cell biological methods in C1QTNF9B research.

What experimental designs best assess the anti-inflammatory properties of C1QTNF9B?

Investigating C1QTNF9B's anti-inflammatory effects requires systematic approaches:

• In vitro inflammation models:

  • Macrophage polarization assays (M1 vs. M2 markers)

  • Cytokine production measurement (TNF-α, IL-1β, IL-6)

  • NF-κB pathway activation assessment

  • Treat cells with recombinant C1QTNF9B (1-5 μg/mL)

• In vivo inflammation assessment:

  • Leukocyte infiltration quantification by immunofluorescence with CD45 antibodies

  • Flow cytometry analysis of cardiac tissue for immune cell subpopulations

  • Cytokine/chemokine profiling in tissue lysates

  • Compare CTRP9B knockout vs. overexpression models

• Transcriptomic analysis:

  • RNA sequencing of cardiac tissue from wild-type and C1QTNF9B-modulated mice

  • Focus on inflammatory gene signatures

  • Key pathways: NF-κB, JAK-STAT, inflammasome activation

• Mechanistic investigations:

  • Receptor identification for anti-inflammatory effects

  • Signaling pathway delineation

  • Direct binding partners in immune cells

• Translation to human studies:

  • Correlation of circulating C1QTNF9B with inflammatory markers

  • Ex vivo studies using human samples

  • Genetic association studies for inflammatory conditions

These approaches comprehensively evaluate the anti-inflammatory properties of C1QTNF9B and identify underlying mechanisms for potential therapeutic development.