C9orf156 Antibody

What is C9orf156 protein and what is its physiological function?

C9orf156, also known as Nef-associated protein 1 (NAP1) or thioesterase NAP1, is a 441 amino acid protein that is ubiquitously expressed throughout human tissues. The protein functions primarily as an enzyme that hydrolyzes acyl-CoA thioesters . C9orf156 belongs to the UPF0066 (virR) family and is encoded by a gene that maps to human chromosome 9q22.33 . The protein has a molecular weight of approximately 49 kDa as calculated from its amino acid sequence .

The protein sequence of C9orf156 (UniProt ID: Q9BU70) shows conservation across various species, suggesting important biological functions that have been maintained throughout evolution. Research indicates potential roles in cellular metabolism through its thioesterase activity, but detailed mechanistic studies are still emerging in the literature.

What applications are suitable for C9orf156 antibodies?

C9orf156 antibodies have been validated for multiple experimental applications in molecular and cellular biology research. According to product documentation, the following applications have been confirmed:

The optimal dilutions should be determined by each laboratory based on their specific experimental conditions and sample types . When establishing protocols, researchers should include appropriate positive and negative controls, particularly utilizing cell lines with known C9orf156 expression levels, and ideally, knockout controls for definitive validation.

What species reactivity has been confirmed for C9orf156 antibodies?

The cross-species reactivity prediction is based primarily on sequence alignment of the immunogen used for antibody production. Species with high sequence conservation in the epitope region are more likely to show cross-reactivity. When working with species other than human or rat, preliminary validation experiments are strongly recommended.

What are the recommended methods for validating C9orf156 antibodies?

Rigorous antibody validation is critical for ensuring experimental reproducibility when working with C9orf156 antibodies. A recommended validation pipeline includes:

• Cell line selection: Utilize the PaxDB database (https://pax-db.org/) to identify cell lines with high endogenous expression of C9orf156 .

• Knockout control generation: Develop CRISPR/Cas9-mediated knockout (KO) cell lines of C9orf156 in the selected high-expression cell line .

• Immunoblot screening: Test antibodies by comparing signal between parental and KO cell lines to confirm specificity .

• Quantitative expression profiling: Use validated antibodies to identify optimal cell lines with highest C9orf156 expression for future experiments .

• Multi-application validation: Screen validated antibodies in additional applications such as immunoprecipitation and immunofluorescence using the generated knockout controls .

This comprehensive validation approach helps ensure that experimental results truly reflect C9orf156 biology rather than non-specific interactions or artifacts.

What is known about C9orf156's potential role in disease pathways?

Research has identified potential associations between C9orf156 and certain disease pathways:

• Multiple Sclerosis (MS): Genetic studies have identified single nucleotide polymorphism (SNP) rs7855251, which is associated with C9orf156 and TRIM14, as potentially linked to MS susceptibility . This suggests a possible role for C9orf156 in inflammatory or autoimmune processes.

• Chromosome 9-related disorders: Since C9orf156 is located on chromosome 9q22.33, it may have relevance to disorders associated with this chromosomal region . Chromosome 9 encompasses significant interferon gene clusters and has been implicated in various genetic disorders.

The precise mechanistic role of C9orf156 in these disease processes remains to be fully elucidated. Researchers investigating these disease associations should exercise caution when interpreting results and implement thorough controls to distinguish correlation from causation.

What challenges exist in detecting endogenous C9orf156 expression?

Detection of endogenous C9orf156 presents several challenges researchers should consider:

• Expression level variation: According to proteomic database analyses, C9orf156 is expressed at relatively low levels in most tissues and cell lines, which can make detection challenging using standard protocols .

• Antibody specificity concerns: As demonstrated with other proteins like C9ORF72, antibodies that cannot actually recognize the target protein have been used in published research, raising concerns about data interpretation . This emphasizes the critical importance of proper antibody validation.

• Splice variant detection: Different splice variants of C9orf156 may exist, potentially affecting epitope availability and antibody recognition.

To address these challenges, researchers should:

• Use cell lines with relatively high expression as positive controls

• Implement knockout or knockdown controls

• Consider signal amplification techniques for low-abundance detection

• Validate results using multiple antibodies recognizing different epitopes where possible

What controls should be implemented when using C9orf156 antibodies?

Appropriate controls are essential for generating reliable data with C9orf156 antibodies:

Essential controls include:

• Positive control: Cell lines or tissues with confirmed high expression of C9orf156 (based on proteomics data or validated by other methods).

• Negative control: Ideally, a CRISPR/Cas9-generated knockout of C9orf156 in the same cell line used as a positive control . Alternative negative controls include:

  • siRNA or shRNA knockdown samples (e.g., using C9orf156 shRNA lentiviral particles)

  • Cell lines with naturally low or absent expression (verified by multiple methods)

• Technical controls:

  • Primary antibody omission control

  • Isotype control (using a non-specific antibody of the same isotype)

  • Blocking peptide competition assay (when available)

The most rigorous validation comes from comparing signals between parental and knockout cell lines, as recommended in advanced antibody validation protocols .

What optimization strategies improve C9orf156 detection in immunoblotting?

For optimal detection of C9orf156 in Western blotting applications, consider the following optimization strategies:

• Sample preparation:

  • Use freshly prepared cell lysates when possible

  • Include protease inhibitors to prevent degradation

  • Consider membrane enrichment protocols if studying membrane-associated fractions

• Electrophoresis and transfer conditions:

  • C9orf156 has a calculated molecular weight of 49 kDa ; use appropriate percentage gels (10-12%)

  • Optimize transfer conditions for proteins in this size range

  • Consider wet transfer for more consistent results

• Antibody incubation:

  • Perform a dilution series to determine optimal antibody concentration

  • Test both short (1-2 hours) and overnight primary antibody incubations at different temperatures

  • Use appropriate blocking buffers to minimize background

• Detection system:

  • For low abundance targets, consider using high-sensitivity detection systems

  • Compare chemiluminescence vs. fluorescence-based detection systems

• Signal validation:

  • Confirm band specificity using knockout controls

  • Verify expected molecular weight (approximately 49 kDa for C9orf156)

How can C9orf156 antibodies be effectively used in immunohistochemistry and immunofluorescence?

For successful detection of C9orf156 in tissue and cellular imaging applications:

• Fixation optimization:

  • Compare different fixation methods (paraformaldehyde, methanol, acetone)

  • Optimize fixation duration to balance epitope preservation and cellular morphology

• Antigen retrieval:

  • Test multiple antigen retrieval methods (heat-induced with citrate buffer, EDTA, or enzymatic methods)

  • Optimize retrieval duration and temperature

• Antibody conditions:

  • Determine optimal antibody dilution through titration experiments

  • Test both short (1-2 hours) and overnight incubations

  • Compare room temperature vs. 4°C incubation

• Signal amplification:

  • For low-abundance targets, consider tyramide signal amplification or other amplification systems

  • Evaluate signal-to-noise ratio with different detection systems

• Controls and validation:

  • Include positive and negative controls in each experiment

  • Perform peptide competition assays when available

  • Compare localization patterns with published literature and protein database information

How can researchers address non-specific binding with C9orf156 antibodies?

Non-specific binding is a common challenge in antibody-based applications. For C9orf156 antibodies, consider these troubleshooting approaches:

• Antibody validation:

  • Verify antibody specificity using knockout controls as described in modern validation pipelines

  • Be skeptical of results from non-validated antibodies, as previous research has shown cases where widely-used antibodies failed to recognize their intended targets

• Blocking optimization:

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

  • Increase blocking duration or concentration if background remains high

• Antibody dilution:

  • Increase antibody dilution to reduce non-specific binding

  • Consider longer incubation times with more dilute antibody solutions

• Washing protocols:

  • Increase washing duration and/or number of washes

  • Test different detergent concentrations in wash buffers

• Pre-adsorption:

  • For tissue work with high background, consider pre-adsorbing the antibody with tissue powder

• Alternative antibodies:

  • If persistent non-specific binding occurs, test antibodies from different manufacturers or those recognizing different epitopes

What approaches can improve reproducibility in C9orf156 research?

To enhance research reproducibility when working with C9orf156 antibodies:

• Standardized reporting:

  • Document complete antibody information (manufacturer, catalog number, lot number, RRID)

  • Use proper citation format (e.g., "Affinity Biosciences Cat# DF2619, RRID:AB_2839825")

• Multi-antibody verification:

  • Validate key findings using multiple antibodies recognizing different epitopes

  • Compare monoclonal and polyclonal antibodies when available

• Complementary methods:

  • Confirm protein expression using orthogonal approaches (mRNA detection, mass spectrometry)

  • Use genetic approaches (overexpression, knockdown, knockout) to validate functionality

• Rigorous controls:

  • Always include appropriate positive and negative controls

  • Use genetic manipulation (CRISPR/Cas9 knockout or shRNA knockdown) for definitive validation

• Protocol standardization:

  • Develop and adhere to standard operating procedures within research groups

  • Document all experimental conditions meticulously

What emerging applications might benefit from C9orf156 antibody research?

As research on C9orf156 continues to evolve, several promising research directions emerge:

• Functional characterization:

  • Further elucidation of the thioesterase activity and its biological significance

  • Identification of specific acyl-CoA substrates and their metabolic pathways

• Disease associations:

  • Investigation of the potential role in Multiple Sclerosis, given the genetic association identified in SNP studies

  • Exploration of interaction with other proteins implicated in neurological disorders

• Intracellular localization:

  • Detailed mapping of C9orf156 subcellular localization using high-resolution microscopy

  • Co-localization studies with organelle markers to determine functional compartmentalization

• Interactome mapping:

  • Identification of C9orf156 binding partners through immunoprecipitation studies

  • Characterization of protein complexes involving C9orf156

• Therapeutic targeting:

  • Assessment of C9orf156 as a potential biomarker in diseases with identified genetic associations

  • Evaluation as a possible therapeutic target in relevant pathways

Researchers pursuing these directions should implement the rigorous antibody validation methods described previously to ensure reliable and reproducible findings.