C20orf27 Antibody

What is C20orf27 and what are its key biological functions?

C20orf27 is a reported synonym of the ADISSP gene, which encodes the adipose secreted signaling protein. This protein functions in carbohydrate metabolism and homeostasis, among other biological roles. The human version of C20orf27 has a canonical amino acid length of 174 residues and a protein mass of 19.3 kilodaltons, with two identified isoforms . It is characterized as a secreted protein and a member of the ADISSP protein family .

Recent research has demonstrated that C20orf27 serves as a novel bona fide PP1 regulatory protein, representing a significant fraction (>5%) of the total pool of PP1cat regulatory proteins captured from HeLa/BAC cell lines . Additionally, studies have revealed that C20orf27 plays a crucial role in promoting cell growth and proliferation in colorectal cancer by activating the TGFβR-TAK1-NFĸB pathway through its interaction with PP1c (the catalytic subunit of type 1 phosphatase) .

What applications are most suitable for C20orf27 antibodies?

C20orf27 antibodies are versatile tools that can be employed in multiple experimental techniques. Based on current research applications, the following methods have proven effective:

When selecting an application, consider the nature of your research question and the level of sensitivity and specificity required. For quantitative studies of expression levels, Western blot or ELISA are recommended, while localization studies benefit from IHC or IF approaches .

How should I validate C20orf27 antibody specificity for my experiments?

Thorough validation of C20orf27 antibodies is critical for generating reliable research data. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines or tissues with known C20orf27 expression levels. Colorectal cancer cell lines such as SW480 and HT29 have been documented to express high levels of C20orf27, while HCT15 and DLD-1 cells express lower levels . Normal intestinal epithelial cells (NCM460) can serve as baseline expression controls .

• Knockout/knockdown verification: Employ CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of C20orf27 to confirm antibody specificity. The change in signal intensity should correlate with the degree of knockdown .

• Peptide competition assay: Pre-incubate the antibody with excess purified C20orf27 protein or immunizing peptide before application to your samples. A specific antibody will show reduced or absent staining.

• Multiple antibody comparison: Use antibodies raised against different epitopes of C20orf27 to confirm consistent staining patterns and expression levels.

• Cross-reactivity assessment: Especially important for polyclonal antibodies, verify the absence of non-specific binding to related proteins in the ADISSP family.

Documentation of these validation steps is essential for publication and reproducibility of your findings.

What are the optimal protocols for detecting C20orf27 via Western blot?

Optimizing Western blot protocols for C20orf27 detection requires attention to several critical parameters:

• Sample preparation:

  • Use RIPA buffer supplemented with protease and phosphatase inhibitors

  • Sonicate briefly to shear DNA and reduce sample viscosity

  • Centrifuge at 14,000g for 15 minutes at 4°C to remove debris

• Protein loading:

  • Load 20-50 μg of total protein per well

  • Include positive controls (e.g., SW480 or HT29 cell lysates)

• Gel electrophoresis:

  • Use 12-15% SDS-PAGE gels due to C20orf27's relatively small size (19.3 kDa)

  • Run at 100V until the dye front reaches the bottom of the gel

• Transfer conditions:

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C

  • Use 0.2 μm pore size PVDF for better retention of small proteins

• Blocking:

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

• Antibody incubation:

  • Primary antibody: Dilute according to manufacturer's recommendation (typically 1:500-1:2000) in blocking buffer; incubate overnight at 4°C

  • Secondary antibody: Use HRP-conjugated antibodies at 1:5000-1:10000 dilution for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) substrates

  • Expose membrane for various durations to capture optimal signal

Remember that the expected molecular weight of C20orf27 is approximately 19.3 kDa, but post-translational modifications or the presence of isoforms may result in additional bands .

How can I troubleshoot poor C20orf27 antibody performance in immunohistochemistry?

When facing suboptimal results with C20orf27 antibodies in immunohistochemistry, systematically address these common issues:

• Weak or absent signal:

  • Optimize antigen retrieval methods (try both citrate buffer pH 6.0 and EDTA buffer pH 9.0)

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Ensure tissue fixation was appropriate (overfixation can mask epitopes)

• High background staining:

  • Increase blocking time or blocking agent concentration

  • Optimize antibody dilution (test serial dilutions)

  • Include additional washing steps

  • Use more specific secondary antibody

  • Pre-absorb primary antibody with non-specific proteins

• Non-specific staining:

  • Validate antibody specificity using knockdown controls

  • Include isotype control antibodies

  • Perform peptide competition assay

  • Use monoclonal antibodies for higher specificity

• Inconsistent results between experiments:

  • Standardize tissue processing and fixation times

  • Use automated staining platforms if available

  • Prepare fresh reagents for each experiment

  • Include positive control tissues in each batch

• Tissue-specific issues:

  • For colorectal tissue, consider short (15-min) enzymatic pre-treatment with proteinase K

  • Adjust fixation protocols for fatty tissues containing adipocytes where C20orf27/ADISSP may be expressed

Document all optimization steps methodically to establish a reproducible protocol for future experiments.

How can I design experiments to investigate C20orf27's role in the PP1 regulatory network?

Investigating C20orf27's role in the PP1 regulatory network requires a multi-faceted experimental approach:

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation (Co-IP) with antibodies against C20orf27 and PP1c to confirm direct interaction

  • Use proximity ligation assay (PLA) to visualize and quantify interactions in situ

  • Employ yeast two-hybrid or mammalian two-hybrid assays to map interaction domains

  • Validate interactions using purified recombinant proteins and surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

• PP1 holoenzyme complex analysis:

  • Use size exclusion chromatography combined with mass spectrometry to characterize PP1-C20orf27 complexes

  • Perform blue native PAGE to preserve protein complexes for analysis

  • Implement affinity purification coupled with mass spectrometry (AP-MS) to identify additional components of the complex

• Functional studies:

  • Develop phosphatase activity assays to determine how C20orf27 modulates PP1 catalytic activity

  • Generate phospho-mimetic and phospho-dead mutants of C20orf27 to study the effect of its phosphorylation on PP1 regulation

  • Create domain deletion constructs to identify regions critical for PP1 binding and regulation

• Cellular pathway analysis:

  • Use PP1-specific inhibitors (e.g., okadaic acid at low concentrations) to distinguish PP1-dependent effects from other phosphatases

  • Perform RNA-seq or phospho-proteomics after C20orf27 manipulation to identify downstream pathways affected by C20orf27-PP1 interaction

Current research has established that C20orf27 represents more than 5% of the total pool of PP1cat regulatory proteins captured from HeLa/BAC cell lines, indicating its significant role in PP1 regulation .

What methodological approaches are optimal for studying C20orf27's role in colorectal cancer progression?

Given the established role of C20orf27 in promoting colorectal cancer growth through the TGFβR-TAK1-NFĸB pathway , several methodological approaches can be employed:

• Expression analysis in clinical samples:

  • Perform IHC on tissue microarrays containing paired tumor and adjacent normal tissues

  • Correlate C20orf27 expression with clinicopathological parameters and patient outcomes

  • Use laser capture microdissection to isolate specific cell populations for more precise analysis

• Functional validation in cell models:

  • Generate stable C20orf27 overexpression and knockdown cell lines using lentiviral vectors

  • Assess cell proliferation using multiple methods (WST-1 assays, colony formation, EdU incorporation)

  • Analyze cell cycle progression using flow cytometry and markers like CyclinD1

  • Evaluate apoptosis markers (Bcl-2, Bax, cleaved-caspase3) in response to C20orf27 modulation

• Pathway analysis:

  • Monitor NFĸB pathway activation through:

    • Western blot for phosphorylated IĸB and phosphorylated p65

    • NFĸB reporter assays

    • Chromatin immunoprecipitation (ChIP) to identify NFĸB binding to target genes

  • Use specific inhibitors of the TGFβR-TAK1-NFĸB pathway to confirm mechanistic involvement

  • Perform rescue experiments with constitutively active or dominant-negative pathway components

• In vivo validation:

  • Establish xenograft models using C20orf27-modulated cell lines

  • Evaluate tumor growth, invasion, and metastasis

  • Test pathway-specific inhibitors in combination with C20orf27 targeting

Research has demonstrated that C20orf27 overexpression increases cell mitochondrial dehydrogenase activity and colony formation in HCT15 and DLD-1 cells, while silencing C20orf27 in SW480 and HT29 cells inhibits these activities . These findings provide a foundation for further mechanistic studies.

How can advanced microscopy techniques be applied to study C20orf27 subcellular localization and dynamics?

Advanced microscopy approaches offer powerful insights into C20orf27 biology beyond conventional immunofluorescence:

• Super-resolution microscopy:

  • Use Structured Illumination Microscopy (SIM) to achieve ~120 nm resolution

  • Apply Stimulated Emission Depletion (STED) microscopy for ~50 nm resolution

  • Implement Single-Molecule Localization Microscopy (SMLM) techniques such as PALM or STORM for ~20 nm resolution

  • These techniques can reveal previously undetectable co-localization with PP1c or other interaction partners

• Live-cell imaging:

  • Generate fluorescent protein fusions (e.g., C20orf27-GFP) to monitor dynamics in real-time

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure mobility and binding kinetics

  • Employ FRET (Förster Resonance Energy Transfer) or BiFC (Bimolecular Fluorescence Complementation) to visualize protein-protein interactions with PP1c in living cells

• Correlative microscopy:

  • Combine light and electron microscopy (CLEM) to correlate fluorescence signals with ultrastructural context

  • Use immunogold labeling for transmission electron microscopy to precisely localize C20orf27 at the ultrastructural level

• Spatial multi-omics:

  • Apply proximity labeling techniques (BioID, APEX) with C20orf27 as the bait to identify proteins in its immediate vicinity

  • Use imaging mass cytometry or multiplexed ion beam imaging to simultaneously visualize multiple proteins in the C20orf27 interactome

• Quantitative image analysis:

  • Implement machine learning algorithms for automated detection of C20orf27 localization patterns

  • Use computational modeling to predict dynamic changes in response to stimuli

These approaches can help determine whether C20orf27's subcellular distribution changes in response to cellular stresses, cell cycle progression, or in pathological conditions such as colorectal cancer, providing deeper insights into its functional roles.

What are the considerations for developing and applying phospho-specific C20orf27 antibodies?

Developing phospho-specific antibodies for C20orf27 requires specialized approaches and careful validation:

• Identification of key phosphorylation sites:

  • Perform mass spectrometry-based phospho-proteomics to identify endogenous phosphorylation sites

  • Use bioinformatic tools to predict likely phosphorylation sites based on consensus sequences

  • Prioritize sites that are evolutionarily conserved or located in functional domains

  • Consider sites that might be regulated during PP1 interaction or NFĸB pathway activation

• Antibody generation strategy:

  • Design phospho-peptides (typically 10-15 amino acids) centered around the phosphorylation site

  • Include a C- or N-terminal cysteine for conjugation to carrier protein

  • Immunize rabbits or other suitable host species

  • Consider using phospho-specific monoclonal antibody development for highest specificity

• Rigorous validation requirements:

  • Test antibody against phosphorylated and non-phosphorylated peptides by ELISA

  • Validate using wild-type protein versus phospho-null mutants (S/T→A or Y→F)

  • Confirm specificity with lambda phosphatase treatment of samples

  • Validate in cells treated with kinase inhibitors or activators affecting the relevant pathway

• Applications in research:

  • Monitor C20orf27 phosphorylation status during cell cycle progression

  • Assess changes in phosphorylation in response to growth factors or stress

  • Investigate how phosphorylation affects interaction with PP1c

  • Map phosphorylation dynamics during colorectal cancer progression

• Technical considerations for use:

  • Include phosphatase inhibitors in all buffer preparations

  • Optimize sample handling to minimize ex vivo dephosphorylation

  • Consider enrichment strategies for phosphorylated proteins prior to analysis

Phospho-specific antibodies would be particularly valuable for investigating whether C20orf27's role in the TGFβR-TAK1-NFĸB pathway is regulated by its own phosphorylation status, potentially revealing additional layers of control in colorectal cancer progression.

How can CRISPR/Cas9 technology be utilized for studying C20orf27 function?

CRISPR/Cas9 technology offers versatile approaches for dissecting C20orf27 function:

• Gene knockout strategies:

  • Design guide RNAs targeting early exons of C20orf27 using established design tools

  • Create complete knockout cell lines to study loss-of-function phenotypes

  • Generate conditional knockouts (using Cre-loxP or inducible systems) to study temporal aspects of C20orf27 function

  • Develop tissue-specific knockouts in mouse models to study organ-specific roles

• Domain-specific modifications:

  • Use homology-directed repair (HDR) to introduce specific mutations in functional domains

  • Create truncation mutants to study domain-specific functions

  • Generate epitope-tagged versions of endogenous C20orf27 for improved antibody detection

• Regulatory element editing:

  • Target promoter or enhancer regions to study transcriptional regulation

  • Use CRISPRi (dCas9-KRAB) to repress transcription without altering the genomic sequence

  • Employ CRISPRa (dCas9-activators) to upregulate endogenous C20orf27 expression

• High-throughput screening:

  • Conduct CRISPR screens to identify genes that synthetically interact with C20orf27

  • Use CRISPR activation/inhibition screens to identify pathways that modulate C20orf27-dependent phenotypes

  • Implement base editing or prime editing for precise nucleotide changes

• Single-cell analysis:

  • Combine CRISPR editing with single-cell RNA-seq to study cell-specific responses

  • Use lineage tracing approaches to follow the fate of C20orf27-modified cells in complex tissues

The C20orf27 CRISPR guide RNA sequences designed by the laboratory of Feng Zhang at the Broad Institute have been optimized to efficiently target the C20orf27 gene with minimal risk of off-target Cas9 binding elsewhere in the genome . These validated guide RNAs provide a solid starting point for designing C20orf27 knockout experiments.

What controls should be included when studying C20orf27 in cancer models?

Rigorous control selection is critical for valid interpretation of C20orf27's role in cancer:

• Cell line controls:

  • Include multiple cell lines with varying endogenous C20orf27 expression levels

  • Use matched normal and cancer cell lines (e.g., NCM460 normal intestinal epithelial cells vs. colorectal cancer cell lines)

  • Consider patient-derived primary cells alongside established cell lines

  • Use isogenic cell lines differing only in C20orf27 status to minimize confounding variables

• Genetic modification controls:

  • For overexpression: include empty vector controls and wild-type C20orf27

  • For knockdown: use non-targeting siRNA/shRNA and multiple independent C20orf27-targeting constructs

  • For CRISPR editing: include non-targeting gRNA controls and verify outcomes with multiple gRNAs

  • Generate rescue cell lines re-expressing C20orf27 in knockout backgrounds

• Pathway analysis controls:

  • Use positive controls known to activate the TGFβR-TAK1-NFĸB pathway

  • Include appropriate inhibitors (e.g., NFĸB inhibitors) as negative controls

  • Perform parallel analyses of known pathway components (e.g., p65, IĸB) to validate effects

• Experimental validation controls:

  • Perform parallel assays measuring the same phenotype (e.g., both WST-1 and colony formation for proliferation)

  • Include time course analyses to distinguish primary from secondary effects

  • Validate in vivo findings with ex vivo analyses of the same specimens

Research has shown that C20orf27's expression in colorectal cancer tissues is significantly higher than in adjacent normal tissues, providing a foundation for comparative studies .

How can I reconcile conflicting data when studying C20orf27 across different experimental systems?

Addressing contradictory results requires systematic evaluation:

• Biological context variations:

  • Cell type differences: C20orf27 may function differently in various cellular contexts

  • Species differences: Human and mouse C20orf27 may have distinct functions or regulation

  • Tissue microenvironment: In vitro vs. in vivo conditions may affect C20orf27 activity

  • Disease stage: Early vs. late cancer stages may show different C20orf27 dependencies

• Technical considerations:

  • Antibody differences: Various antibodies may recognize different epitopes or isoforms

  • Expression level artifacts: Overexpression may cause non-physiological effects

  • Temporal factors: Acute vs. chronic manipulation may yield different outcomes

  • Assay sensitivity: Different detection methods have varying sensitivity thresholds

• Resolution strategies:

  • Perform dose-response experiments to identify threshold effects

  • Use multiple independent methodologies to verify findings

  • Conduct time course analyses to capture dynamic changes

  • Implement unbiased systematic approaches (e.g., proteomics, transcriptomics)

  • Evaluate post-translational modifications that might explain context-specific effects

• Integrative analysis:

  • Apply computational modeling to integrate disparate datasets

  • Use machine learning approaches to identify patterns in complex data

  • Implement Bayesian networks to infer causal relationships

  • Evaluate genetic background effects that might explain differential responses

When faced with contradictory results, consider that C20orf27's dual role as both a PP1 regulatory protein and an activator of the TGFβR-TAK1-NFĸB pathway may reflect context-dependent functions that vary across experimental systems.

What experimental designs are most effective for translating C20orf27 findings to clinical applications?

Translational research on C20orf27 requires carefully designed experiments that bridge basic and clinical science:

• Patient sample analyses:

  • Conduct large-scale IHC studies on tissue microarrays with comprehensive clinical data

  • Perform retrospective analyses correlating C20orf27 expression with treatment response

  • Use liquid biopsies to evaluate circulating C20orf27 as a potential biomarker

  • Integrate genomic, transcriptomic, and proteomic data from patient cohorts

• Preclinical model development:

  • Generate patient-derived xenografts (PDXs) that maintain tumor heterogeneity

  • Develop genetically engineered mouse models (GEMMs) with tissue-specific C20orf27 alterations

  • Use organoid cultures from normal and cancer tissues to study C20orf27 function

  • Implement humanized mouse models for immuno-oncology studies

• Therapeutic target validation:

  • Conduct synthetic lethality screens to identify vulnerabilities in C20orf27-high cancers

  • Perform drug sensitivity profiling based on C20orf27 status

  • Develop C20orf27 targeting strategies (e.g., degraders, activity modulators)

  • Test combination strategies targeting C20orf27 and interacting pathways

• Biomarker development pipeline:

  • Establish standardized IHC protocols for clinical C20orf27 detection

  • Develop and validate ELISA or other quantitative assays for C20orf27 in biofluids

  • Create multiplex panels including C20orf27 and related pathway markers

  • Conduct prospective studies to validate C20orf27 as a predictive or prognostic marker

Research has demonstrated that C20orf27 promotes tumor growth in vivo in subcutaneous colorectal cancer models , providing a foundation for further translational studies in preclinical models.

How can I optimize sample preparation for C20orf27 detection in different biological specimens?

Effective C20orf27 detection requires specimen-specific optimization:

• Cell lysate preparation:

  • Use RIPA buffer for general applications, supplemented with protease and phosphatase inhibitors

  • For studying protein interactions, consider gentler lysis buffers (NP-40 or digitonin-based)

  • Include brief sonication (3-5 pulses) to shear DNA without damaging proteins

  • Maintain cold temperature throughout processing to preserve protein integrity

• Tissue processing:

  • For fresh tissues: snap-freeze in liquid nitrogen and store at -80°C

  • For FFPE samples: limit fixation time to 24 hours to preserve antigenicity

  • For frozen sections: use OCT compound and quick-freeze on dry ice

  • Consider using PAXgene or similar fixatives that better preserve protein phosphorylation states

• Subcellular fractionation:

  • Implement differential centrifugation protocols to separate cytosolic, nuclear, and membrane fractions

  • Use density gradient centrifugation for more refined organelle separation

  • Verify fraction purity using established markers (e.g., GAPDH for cytosol, Lamin B1 for nucleus)

  • Use specialized extraction buffers for secreted proteins in culture media

• Protein enrichment strategies:

  • Consider immunoprecipitation to concentrate C20orf27 from dilute samples

  • Use phospho-peptide enrichment (TiO₂ or IMAC) for phosphorylation studies

  • Apply size exclusion chromatography to study C20orf27 in protein complexes

  • Implement affinity purification for studying specific binding partners

• Specialized sample types:

  • For serum/plasma: use protease inhibitors and process quickly to prevent degradation

  • For cerebrospinal fluid: concentrate proteins via precipitation (TCA or acetone)

  • For urine: normalize to creatinine levels and concentrate proteins

  • For exosomes: isolate via ultracentrifugation or commercial kits before analysis

Careful consideration of sample preparation is particularly important when studying C20orf27 in its reported role as a secreted protein , where preservation of native structure may be critical for detection.

What quantitative approaches can accurately measure C20orf27 expression levels?

Accurate quantification of C20orf27 requires selecting appropriate methodologies:

• Protein-level quantification:

  • Western blot: Use densitometry with standard curves for semi-quantitative analysis

  • ELISA: Develop sandwich ELISA for absolute quantification in solution

  • Multiplex protein assays: Include C20orf27 in multiplex bead-based assays

  • Mass spectrometry: Implement targeted proteomics (MRM/PRM) with heavy-labeled standards

• mRNA-level quantification:

  • RT-qPCR: Design exon-spanning primers to distinguish isoforms

  • Digital PCR: Use for absolute quantification without standard curves

  • RNA-seq: Apply for genome-wide expression context

  • NanoString: Consider for direct digital counting without amplification

• Cell-level quantification:

  • Flow cytometry: Use for single-cell protein quantification in cell suspensions

  • Image cytometry: Apply for adherent cells or tissue sections

  • Single-cell RNA-seq: Implement for cell-specific expression profiling

  • Spatial transcriptomics: Consider for tissue-context expression mapping

• Normalization strategies:

  • For Western blot: Normalize to total protein (Ponceau, REVERT) rather than housekeeping proteins

  • For qPCR: Use multiple reference genes validated for stability in your experimental system

  • For IHC: Implement digital pathology with calibrated intensity measurements

  • For proteomics: Use label-free approaches or isotope labeling for relative quantification

• Data analysis approaches:

  • Apply appropriate statistical tests for your experimental design

  • Use power analysis to determine adequate sample sizes

  • Implement quality control metrics for each quantification method

  • Consider biological relevance of fold-changes versus statistical significance