CALCOCO2 Antibody

What is CALCOCO2 and what are its key biological functions?

CALCOCO2 (Calcium binding and coiled-coil domain 2), also known as NDP52 (Nuclear dot protein 52), is a 446 amino acid multifunctional protein with a molecular weight of approximately 52 kDa . It serves as an important autophagy receptor that plays several critical roles in cellular processes:

• Functions as a xenophagy receptor that mediates intracellular bacterial degradation through autophagy

• Regulates mitophagy (selective autophagy of damaged mitochondria)

• Participates in actin cytoskeleton organization and ruffle formation

• Modulates innate immune responses, particularly type I interferon signaling

• Regulates beta cell function and insulin secretion relevant to type 2 diabetes risk

• Shows both antiviral and proviral functions depending on the virus type

CALCOCO2 contains a C-terminal cargo-binding region with two zinc fingers: a dynamic unconventional zinc finger and a C₂H₂-type zinc finger that specifically recognizes ubiquitin chains, enabling it to target ubiquitinated pathogens for degradation .

How is CALCOCO2 expression distributed in human tissues?

CALCOCO2 is expressed in various human tissues with differential distribution patterns:

The protein is primarily localized in the membrane, nucleus, cytoplasmic vesicles, and cytoplasm of cells . In peripheral blood mononuclear cells, CALCOCO2 is mainly expressed in B cells, where it mediates mitophagy and reduces pro-inflammatory cytokine production following stimulation .

What factors should be considered when selecting a CALCOCO2 antibody for specific research applications?

When selecting a CALCOCO2 antibody for research, consider the following methodological factors:

• Antibody type: Determine whether a monoclonal (e.g., mouse IgG2a) or polyclonal (e.g., rabbit) antibody better suits your experimental needs. Monoclonal antibodies offer higher specificity to a single epitope, while polyclonal antibodies provide broader antigen recognition .

• Species reactivity: Verify the antibody's reactivity with your species of interest. Common reactivity profiles include human, mouse, and rat samples .

• Application compatibility: Select an antibody validated for your specific application:

  • Western Blot (WB): Typically used at dilutions between 1:5000-1:50000

  • Immunohistochemistry (IHC): Typically used at dilutions between 1:50-1:500

  • Immunofluorescence (IF/ICC): Typically used at dilutions between 1:400-1:1600

  • Flow cytometry, ELISA, or immunoprecipitation applications

• Epitope location: Consider whether the antibody targets a specific domain of CALCOCO2 that is relevant to your research question (e.g., zinc finger domains, LIR motif) .

• Conjugation requirements: Determine if you need an unconjugated antibody or one conjugated to a detection tag (HRP, FITC, PE, Alexa Fluor) .

What validation methods should be employed to ensure CALCOCO2 antibody specificity?

To ensure antibody specificity for CALCOCO2, implement these methodological validation approaches:

• Positive controls: Verify reactivity using cells known to express CALCOCO2 (e.g., A549, HeLa, Jurkat, or MDA-MB-231 cells) .

• Knockout/knockdown validation: Confirm specificity by comparing antibody signal between wild-type samples and those with CALCOCO2 knocked out (via CRISPR-Cas9) or knocked down (via siRNA/shRNA) .

• Western blot analysis: Confirm the antibody detects a single band at the expected molecular weight (52 kDa), with potential additional bands for known isoforms (human CALCOCO2 has some isoforms; mouse/rat CALCOCO2 has isoforms with MW 28-40 kDa and 67 kDa) .

• Blocking peptide controls: Use peptides containing the epitope recognized by the antibody to confirm binding specificity by demonstrating signal reduction .

• Multiple antibody comparison: Validate findings using independent antibodies targeting different CALCOCO2 epitopes to ensure consistent results .

What are the optimal protocols for using CALCOCO2 antibodies in Western blot applications?

For optimal Western blot results with CALCOCO2 antibodies, follow these methodological steps:

• Sample preparation:

  • Lyse cells in protein lysate buffer (e.g., 100 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 6.8), 10 mM EDTA, and 4% SDS)

  • Include protease inhibitors to prevent protein degradation

  • Quantify protein using BCA or Bradford assay

• Gel electrophoresis and transfer:

  • Use 10-12.5% SDS-PAGE gels for optimal separation

  • Load 20-30 μg of total protein per lane

  • Transfer to PVDF membrane at 100V for 1-2 hours or 30V overnight

• Antibody incubation:

  • Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary CALCOCO2 antibody as recommended (typically 1:5000-1:50000)

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST

  • Incubate with appropriate secondary antibody (e.g., goat anti-mouse IgG, 1:5000) for 1-3 hours at room temperature

  • Wash 3-5 times with TBST

• Detection and analysis:

  • Develop using enhanced chemiluminescence (ECL) reagent

  • Expect bands at approximately 52 kDa for human CALCOCO2

  • Mouse/rat samples may show additional isoforms (28-40 kDa and 67 kDa)

How should CALCOCO2 antibodies be utilized in immunofluorescence experiments to visualize autophagy processes?

For effective immunofluorescence visualization of CALCOCO2 in autophagy processes:

• Cell preparation:

  • Culture cells on coverslips or in chamber slides

  • For autophagy studies, consider treatments that induce or inhibit autophagy (e.g., starvation, bafilomycin A1)

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Antibody staining:

  • Block with 1-5% BSA in PBS for 30-60 minutes

  • Dilute CALCOCO2 antibody optimally (typically 1:400-1:1600)

  • Co-stain with autophagy markers (e.g., LC3, LAMP2, ubiquitin) to visualize colocalization

  • For mitophagy studies, include mitochondrial markers (e.g., TOMM20, MitoTracker)

  • Include DAPI for nuclear staining

• Visualization parameters:

  • CALCOCO2 typically shows cytoplasmic punctate staining during active autophagy

  • In infected cells, look for colocalization with pathogens for xenophagy studies

  • For mitophagy assessment, look for CALCOCO2 colocalization with damaged mitochondria

• Controls and validation:

  • Include CALCOCO2 knockdown/knockout cells as negative controls

  • Use Jurkat cells as positive controls, which have been validated for CALCOCO2 immunofluorescence

  • Consider dual immunofluorescence with two different CALCOCO2 antibodies recognizing different epitopes for validation

How can CALCOCO2 antibodies be employed to investigate viral infection mechanisms?

CALCOCO2 plays complex roles in viral infections, and antibodies can be used to investigate these mechanisms through:

• Co-immunoprecipitation studies:

  • Use CALCOCO2 antibodies for immunoprecipitation followed by detection of viral proteins

  • This approach has revealed interactions between CALCOCO2 and viral proteins such as:

    • Coxsackievirus B3 (CVB3) capsid protein VP1

    • Influenza virus protein PB1-F2

    • Chikungunya virus NSP2 protein

    • Bovine viral diarrhea virus (BVDV) Npro protein

• Temporal expression analysis:

  • Monitor CALCOCO2 levels during viral infection time course

  • Investigate viral protease-mediated cleavage of CALCOCO2:

    • CVB3 proteinase 3C cleaves CALCOCO2 at glutamine 139, generating a stable C-terminal fragment that retains proviral function

• Functional studies:

  • Combine CALCOCO2 immunolabeling with visualization of:

    • Type I interferon signaling components (MAVS, TBK1)

    • Ubiquitinated viral proteins

    • Autophagic machinery recruitment to viral components

• Mechanistic investigation:

  • For viruses where CALCOCO2 has proviral effects (e.g., CVB3, BVDV), examine:

    • Colocalization with components of IFN signaling pathway

    • CALCOCO2-mediated suppression of IFN-α/IFN-β, MX1, ISG15, and OAS1 expression

  • For viruses where CALCOCO2 has antiviral effects, examine:

    • Direct interaction with ubiquitinated viral proteins

    • Recruitment of autophagic machinery to viral components

What methodological approaches can resolve contradictory findings regarding CALCOCO2's role in disease contexts?

CALCOCO2 shows context-dependent roles in different diseases. To resolve contradictory findings:

• Cell type-specific analysis:

  • Compare CALCOCO2 functions across different cell types using immunohistochemistry and cell-specific markers

  • Consider tissue microarrays to assess expression patterns across multiple tissues simultaneously

  • In PBMC studies, use flow cytometry with CALCOCO2 antibodies to analyze expression across immune cell subsets (particularly B cells)

• Genetic variant analysis:

  • Integrate CALCOCO2 antibody studies with genetic information:

    • The G140E variant near the LC3-interacting region (LIR) motif is protective in multiple sclerosis

    • T2D-associated variants at the CALCOCO2 locus affect insulin secretion

  • Compare antibody-based protein detection between different variant carriers

• Context-dependent role investigation:

  • In cancer studies (prostate cancer, glioma), correlate CALCOCO2 expression with:

    • Cell proliferation and colony formation assays

    • Apoptosis markers (Annexin V, cleaved caspase-3)

    • Cell cycle regulators (cyclin-E1, p53)

  • In infectious disease models, examine:

    • Viral/bacterial load

    • Type I interferon response components

    • Autophagy flux markers

• Pathway-specific experimental design:

  • For autophagy studies: Compare CALCOCO2 levels with and without autophagy inhibitors (bafilomycin A1)

  • For inflammatory response: Measure CALCOCO2 effects on cytokine production in relevant cell types

  • For mitophagy: Assess mitochondrial morphology and function in relation to CALCOCO2 levels

• Methodological triangulation:

  • Combine multiple antibody-based techniques (WB, IF, IHC, flow cytometry)

  • Validate with non-antibody methods (qPCR, CRISPR screens)

  • Use complementary functional assays to validate findings

How can researchers troubleshoot non-specific binding or high background issues with CALCOCO2 antibodies?

To troubleshoot and optimize CALCOCO2 antibody performance:

• Antibody dilution optimization:

  • Establish a dilution series to determine optimal concentration

  • For Western blot: Test dilutions from 1:5000 to 1:50000

  • For IHC: Test dilutions from 1:50 to 1:500

  • For IF/ICC: Test dilutions from 1:400 to 1:1600

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time (1-3 hours) or concentration (3-5%)

  • For tissues with high endogenous biotin, use avidin/biotin blocking kit when using biotinylated secondary antibodies

• Antigen retrieval methods for IHC:

  • For CALCOCO2 IHC, suggested antigen retrieval includes:

    • TE buffer pH 9.0 (primary recommendation)

    • Citrate buffer pH 6.0 (alternative approach)

  • Optimize heating time and temperature

• Cross-reactivity assessment:

  • Verify specificity using CALCOCO2 knockout cells

  • Use blocking peptides containing the immunogen sequence to confirm specificity

  • Check for cross-reactivity with related proteins (other CALCOCO family members)

• Sample preparation considerations:

  • Ensure proper fixation (overfixation can mask epitopes)

  • For frozen sections, optimize fixation time

  • For cell lines, confirm expression of CALCOCO2 (positive controls include A549, HeLa, Jurkat, and MDA-MB-231 cells)

What strategies can improve detection of CALCOCO2 in low-expressing tissues or cell types?

For improved detection of CALCOCO2 in challenging samples:

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) for IHC/IF applications

  • Consider biotin-streptavidin systems for enhanced sensitivity

  • For Western blot, use high-sensitivity ECL substrates or longer exposure times

• Sample enrichment approaches:

  • For cell fractionation studies, isolate relevant cellular compartments (cytoplasmic, nuclear, membrane fractions)

  • For tissue samples, consider laser capture microdissection to isolate specific regions

  • Immunoprecipitate CALCOCO2 before detection to concentrate the protein

• Detection system selection:

  • For Western blot, consider fluorescent secondary antibodies and infrared imaging systems for quantitative analysis

  • For microscopy, use high-sensitivity cameras and appropriate filter sets

  • Consider antibody conjugates (HRP, fluorescent dyes) that match your detection system's optimal sensitivity range

• Protocol modifications:

  • Extend primary antibody incubation time (overnight at 4°C or longer)

  • Increase sample concentration (load more protein for Western blot)

  • Reduce washing stringency while maintaining specificity

  • For fixed tissues, optimize permeabilization to improve antibody access while preserving morphology

• Positive controls:

  • Include tissues/cells known to express high levels of CALCOCO2 (skeletal muscle tissue)

  • Consider recombinant CALCOCO2 as a positive control for Western blot applications

How can CALCOCO2 antibodies contribute to research on type 2 diabetes pathogenesis?

Recent studies have identified CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk . Researchers can utilize CALCOCO2 antibodies to:

• Investigate pancreatic beta cell function:

  • Immunostain pancreatic tissue sections from normal and diabetic subjects

  • Correlate CALCOCO2 expression with insulin content and secretion

  • Examine colocalization with insulin granule markers

• Study autophagy-related mechanisms:

  • Visualize autophagosomes and their relationship to insulin granules

  • Monitor alterations in mitochondrial morphology associated with CALCOCO2 deficiency

  • Assess proinsulin-containing immature granules in relation to CALCOCO2 levels

• Examine genetic variant effects:

  • Compare CALCOCO2 protein expression and localization in carriers of T2D-associated variants

  • Correlate with insulin secretion patterns

  • Investigate differential binding to interaction partners in variant carriers

• Pathway analysis:

  • Study CALCOCO2 interactions with autophagy proteins in beta cells

  • Examine the relationship between CALCOCO2 and mitochondrial function

  • Investigate effects on proinsulin processing and insulin granule biogenesis

• Therapeutic target validation:

  • Test effects of modulating CALCOCO2 levels on insulin secretion

  • Screen for compounds that affect CALCOCO2 function or expression

  • Evaluate CALCOCO2 as a biomarker for beta cell dysfunction in T2D

What is the potential role of CALCOCO2 antibodies in cancer research and therapeutic development?

CALCOCO2 has been identified as an oncogene in prostate cancer and glioma , suggesting several applications for CALCOCO2 antibodies in cancer research:

• Expression profiling in tumors:

  • Compare CALCOCO2 protein levels between cancer and normal tissues

  • Correlate expression with clinical parameters and patient outcomes

  • Develop tissue microarrays to screen multiple cancer types

• Functional studies in cancer models:

  • Monitor changes in CALCOCO2 expression during:

    • Cell proliferation and colony formation

    • Apoptosis (combined with Annexin V and cleaved caspase-3 staining)

    • Cell cycle progression

  • Investigate effects of CALCOCO2 knockdown on cyclin-E1 and p53 protein expression

• Mechanistic investigations:

  • Examine CALCOCO2's role in:

    • Autophagosome assembly

    • Nucleophagy

    • Nucleic acid metabolic processes

  • Study interactions with cancer-relevant proteins:

    • Sequestosome-1 (p62)

    • MAP1LC3B

    • IκB kinase subunit γ and NF-κB pathway components

• Therapeutic development applications:

  • Use as a diagnostic marker in prostate cancer and potentially other cancers

  • Evaluate as a prognostic indicator

  • Develop targeting strategies based on CALCOCO2's role in autophagy

• Translational research:

  • Correlate CALCOCO2 expression with response to therapy

  • Evaluate as a companion diagnostic for treatments targeting autophagy

  • Investigate potential for antibody-drug conjugates targeting CALCOCO2-expressing cancer cells