calcoco1 Antibody

What is CALCOCO1 and why is it important in research?

CALCOCO1 is a 691 amino acid protein that shuttles between the cytoplasm and nucleus, functioning as a coactivator for aryl hydrocarbon and nuclear receptors. Research has identified CALCOCO1 as a key player in autophagy regulation and tumor suppression mechanisms . It's gaining increased attention because:

• It forms a calphoglin complex with PPA1 and PGM1

• It acts as a component of both the androgen signaling pathway and the Wnt/β-catenin signaling pathway

• It has recently been identified as a selective autophagy receptor with a specific role in reticulophagy (selective autophagy of the endoplasmic reticulum)

This multifunctional protein exists as three alternatively spliced isoforms (Q9P1Z2-1, 2, and 3) encoded by genes mapping to human chromosome 12q13.13 and mouse chromosome 15 F3 .

How do I select the appropriate CALCOCO1 antibody for my research application?

Selection should be based on your specific experimental requirements:

• Application compatibility: Verify the antibody has been validated for your application (WB, IHC, IF, ELISA)

• Species reactivity: Ensure reactivity with your experimental model (human, mouse, rat)

• Epitope recognition: Consider which region of CALCOCO1 you need to detect

For studies focusing on specific domains, select antibodies targeting relevant regions (e.g., SKICH domain, CLIR, LIR, or coiled-coil domains) .

What are the optimal conditions for using CALCOCO1 antibodies in Western blotting?

For optimal Western blotting results with CALCOCO1 antibodies:

• Sample preparation:

  • Use fresh samples with protease inhibitors

  • CALCOCO1 has an observed molecular weight of approximately 100 kDa (despite calculated MW of 77 kDa)

• Protocol optimization:

  • Blocking: 5% non-fat milk in TBST is generally effective

  • Primary antibody incubation: Overnight at 4°C for optimal sensitivity

  • Dilution ranges: Start with manufacturer recommendations (e.g., 1:5000-1:50000 for Proteintech 84009-4-RR )

• Positive controls: Include validated positive samples such as:

  • HeLa cells

  • 293T cells

  • MCF-7 cells

  • A-549 cells

  • Mouse lung/heart/thymus tissue

• Detection: Anti-rabbit secondary antibodies with appropriate detection systems (HRP/ECL or fluorescent)

Note that CALCOCO1 expression is regulated by MTOR inhibition and autophagy induction, which might affect detection levels in certain experimental conditions .

How should I optimize immunohistochemistry protocols for CALCOCO1 detection?

For successful IHC with CALCOCO1 antibodies:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Antibody dilution:

  • Start with recommended ranges (e.g., 1:400-1:1600 for Proteintech 84009-4-RR )

  • Titrate to optimize signal-to-noise ratio

• Validated positive tissues:

  • Human: cervical cancer, esophagus cancer tissues

  • Mouse/rat: testis tissue

• Detection systems:

  • DAB (3,3'-diaminobenzidine) detection works well

  • Consider tyramide signal amplification for low-expression tissues

• Controls:

  • Include no-primary-antibody controls

  • Consider using tissues from CALCOCO1 knockout models if available

How does CALCOCO1 expression change during autophagy and how can this be monitored?

CALCOCO1 expression is dynamically regulated during autophagy:

• Expression patterns:

  • CALCOCO1 expression increases significantly in cells with disrupted autophagy genes (ATG7 or ATG3)

  • MTOR inhibition (e.g., with MLN0128) further increases CALCOCO1 levels

  • Bafilomycin A1 (an autophagy inhibitor) affects CALCOCO1 expression

• Experimental design for monitoring:

  • Time-course experiments with autophagy inducers (starvation, rapamycin)

  • Compare expression in autophagy-competent vs. autophagy-deficient cells

  • Co-staining with autophagy markers (LC3, p62/SQSTM1)

• Quantification methods:

  • Western blot densitometry normalized to loading controls

  • Immunofluorescence quantification of nuclear vs. cytoplasmic localization

  • qRT-PCR for transcript level changes

This experimental paradigm can help elucidate CALCOCO1's role in the switch between bulk autophagy and selective autophagy (reticulophagy) .

How should I design experiments to investigate CALCOCO1's interaction with LC3 family members?

To study CALCOCO1's interactions with LC3 family members:

• Co-immunoprecipitation approach:

  • Express CALCOCO1-MYCDDK with HA-tagged LC3 family members

  • Immunoprecipitate with anti-HA antibodies

  • Immunoblot for CALCOCO1-MYCDDK

  • CALCOCO1 shows strongest association with MAP1LC3C

• Immunofluorescence co-localization:

  • Co-transfect cells with tagged CALCOCO1 and LC3 family members

  • Look for overlapping signals in discrete puncta

  • Quantify co-localization coefficients

• Mutagenesis studies:

  • Generate mutations in the conserved LIR motif of CALCOCO1

  • The LIR region is essential for LC3 interaction

  • The non-canonical CLIR domain partially mediates this interaction

• Functional assays:

  • Monitor LC3 lipidation (LC3-I to LC3-II conversion) in CALCOCO1 knockdown cells

  • Assess autophagosome formation using fluorescent LC3 reporters

How can I use CALCOCO1 antibodies to investigate its role in reticulophagy?

Investigating CALCOCO1's role in reticulophagy requires combined approaches:

• ER-specific autophagy assays:

  • ER-tracker™ Red to measure relative ER to cell area ratios

  • Co-staining with ER markers (CANX, SEC61B) and autophagy markers

  • ER-phagy reporter systems (e.g., RTNLA-GFP-mCherry)

• Biochemical approaches:

  • Subcellular fractionation to isolate ER membranes

  • Western blotting for ER proteins (RAB9A, LMAN2) in CALCOCO1 KO vs. control cells

  • Mass spectrometry proteomics to identify additional ER substrates regulated by CALCOCO1

• Genetic manipulation:

  • CRISPR/Cas9-mediated CALCOCO1 knockout

  • Domain-specific mutants to determine which regions are required for reticulophagy

  • Rescue experiments with wild-type vs. mutant CALCOCO1

• Induction conditions:

  • MTOR inhibitors (e.g., MLN0128) to induce reticulophagy

  • ER stress inducers (thapsigargin, tunicamycin) to trigger ER-phagy

Mass spectrometry proteomics reveals that CALCOCO1 knockout leads to an accumulation of select ER-localized proteins (RAB9A, LMAN2), supporting its role in reticulophagy .

What experimental approaches can resolve the apparent contradiction between CALCOCO1's role in tumor suppression versus its positive correlation with tumorigenicity?

This paradox requires sophisticated experimental design:

• Cell-type specific analysis:

  • Compare CALCOCO1 function in different cancer models

  • In breast cancer, CALCOCO1 expression positively correlates with tumorigenicity

  • Yet CALCOCO1 functions in autophagy, which can be tumor-suppressive

• Functional assays:

  • Proliferation assays in CALCOCO1 KO vs. control cells with/without MTOR inhibitors

  • Migration/invasion assays to assess metastatic potential

  • Mammosphere formation assays to evaluate cancer stem cell properties

  • CALCOCO1 KO cells show ~50% fewer mammospheres compared to control cells

• Mechanistic investigations:

  • Determine if CALCOCO1's effects are autophagy-dependent or independent

  • Analyze CALCOCO1's role in specific signaling pathways (Wnt/β-catenin, androgen signaling)

  • Evaluate context-dependent interaction partners

• In vivo models:

  • Xenograft models with CALCOCO1 knockdown/overexpression

  • Patient-derived organoids with varying CALCOCO1 levels

  • Correlation with clinical outcomes in patient samples

This experimental program can help clarify whether CALCOCO1's role is context-dependent, with tumor-promoting or tumor-suppressive effects based on cancer type, stage, or microenvironment.

How can I investigate the potential role of CALCOCO1 in other types of selective autophagy beyond reticulophagy?

Proteomics data suggests CALCOCO1 may mediate additional selective autophagy pathways:

• Mitophagy (mitochondrial autophagy):

  • Monitor mitochondrial proteins (e.g., ATP5MF) in CALCOCO1 KO cells

  • Use mitophagy-specific reporters (mito-Keima, mt-mCherry-GFP)

  • Co-localization of CALCOCO1 with mitochondrial markers during stress

• Pexophagy (peroxisome autophagy):

  • Track peroxisomal proteins (e.g., PECR) in CALCOCO1 KO cells

  • Peroxisome abundance assays using peroxisome-targeted fluorescent proteins

  • Peroxisome proliferator treatments (e.g., clofibrate)

• Ferritinophagy (ferritin degradation):

  • Measure ferritin levels (FTL, FTH1) in CALCOCO1-deficient cells

  • Iron chelation treatments to induce ferritinophagy

  • Transferrin uptake assays

• Experimental approaches:

  • Proximity labeling methods (BioID, APEX) to identify CALCOCO1 interaction partners

  • Live-cell imaging with fluorescently tagged CALCOCO1 and organelle markers

  • Organelle isolation followed by proteomic analysis

Mass spectrometry of MLN-treated HEK293 cells with CRISPR-mediated CALCOCO1 deletion revealed increased levels of mitochondrial, peroxisomal, and ferritin proteins, suggesting CALCOCO1 may mediate multiple forms of selective autophagy .

Why might I observe differential molecular weights for CALCOCO1 in Western blotting experiments?

Several factors can explain molecular weight variations:

• Reported variations:

  • Calculated MW: 77 kDa

  • Observed MW: 100 kDa or 77 kDa depending on the antibody used

• Potential causes:

  • Post-translational modifications (phosphorylation, ubiquitination)

  • Alternative splicing (three known isoforms of CALCOCO1)

  • The presence of tags in recombinant proteins

  • SDS-resistant protein complexes

• Resolution approaches:

  • Use different lysis buffers with varying detergent strengths

  • Include phosphatase inhibitors if phosphorylation is suspected

  • Compare different antibodies recognizing distinct epitopes

  • Consider deglycosylation treatments if glycosylation is suspected

• Validation methods:

  • siRNA/shRNA knockdown to confirm specificity

  • CRISPR knockout cell lysates as negative controls

  • Overexpression of tagged CALCOCO1 as positive controls

These technical considerations are essential for accurate interpretation of Western blotting results when studying CALCOCO1.

What are the most common pitfalls when investigating CALCOCO1's interaction with autophagy pathways?

Key challenges include:

• Expression variability:

  • CALCOCO1 expression is nearly undetectable in some cell lines (e.g., Hep3B)

  • Expression is highly regulated by MTOR and autophagy status

  • Experimental treatment timing is critical

• Functional redundancy:

  • CALCOCO1 belongs to a family with similar domain structure to CALCOCO2 and TAX1BP1

  • Family members may compensate for CALCOCO1 loss

  • Consider multiple knockdown approaches

• Context-dependent effects:

  • CALCOCO1 knockout impairs reticulophagy but increases bulk autophagy

  • Effects may vary by cell type and stress condition

  • Design experiments with appropriate controls for each context

• Technical considerations:

  • Autophagy is a dynamic process requiring time-course analyses

  • Basal vs. induced autophagy should be distinguished

  • Include proper autophagy controls (Bafilomycin A1, chloroquine)