C9orf78 Antibody

What is C9orf78 and why is it studied in research?

C9orf78 (chromosome 9 open reading frame 78) is a 34 kDa protein encoded by the C9orf78 gene (Gene ID: 51759). While the complete function of C9orf78 remains largely unknown, it has become an important research target due to its expression patterns across multiple tissue types . The protein consists of 289 amino acids and has been detected in human, mouse, and rat samples, suggesting evolutionary conservation that may indicate important biological functions . Research interest in C9orf78 continues to grow as scientists investigate its potential roles in cellular processes and potential associations with various physiological and pathological conditions.

How can I determine which cell lines express C9orf78 at high levels?

To identify cell lines with high C9orf78 expression, consult proteomics databases that catalog protein expression across various cell types. Based on available data, HeLa and HepG2 cells have been documented to express detectable levels of C9orf78 . For optimal experimental design, validate expression in your specific cell line of interest using Western blot with a validated antibody such as 14496-1-AP, which has been specifically tested in HeLa and HepG2 cells . This confirmation step is crucial before proceeding with more complex experiments, especially when planning knockdown or knockout studies that rely on baseline expression.

What are the known molecular characteristics of the C9orf78 protein?

C9orf78 has the following molecular characteristics:

The protein's amino acid sequence suggests potential functional domains, though further structural and functional studies are needed to elucidate its precise biological role .

How can I select the most appropriate C9orf78 antibody for my research?

When selecting a C9orf78 antibody, consider these critical factors:

• Application compatibility: Not all antibodies perform equally across different applications. For example, some antibodies may excel in immunoblotting but perform poorly in immunoprecipitation or immunohistochemistry .

• Validation status: Prioritize antibodies validated through knockout (KO) verification, where the antibody has been tested against samples from which the target protein has been genetically deleted. This represents the gold standard for antibody validation .

• Species reactivity: Confirm the antibody's reactivity with your experimental model system. For example, antibody 14496-1-AP has been validated for reactivity with human, mouse, and rat samples .

• Clonality considerations: Consider whether a monoclonal or polyclonal antibody better suits your application. Polyclonal antibodies like 14496-1-AP recognize multiple epitopes and can provide stronger signals, while monoclonal antibodies offer higher specificity for a single epitope .

• Published literature: Review research publications that have successfully employed specific C9orf78 antibodies in applications similar to yours.

What validation methods should I use to confirm the specificity of a C9orf78 antibody?

Implementing a rigorous validation strategy is crucial for ensuring antibody specificity:

• CRISPR/Cas9 knockout controls: The gold standard approach involves generating cell lines with C9orf78 knocked out using CRISPR/Cas9 technology. Compare antibody reactivity between parental and knockout cell lines across your intended applications .

• Multi-application testing: An antibody performing well in one application may fail in others. Test your antibody in all intended applications (Western blot, immunoprecipitation, immunofluorescence) using proper controls .

• Peptide competition assays: Pre-incubate the antibody with excess purified C9orf78 protein or immunizing peptide before application to your experimental samples. Signal reduction indicates specific binding.

• Cross-species reactivity: Evaluate antibody performance across species when working with different model organisms. For example, while some antibodies recognize both human and murine variants of a protein, others may show species-specific reactivity .

• Orthogonal validation: Validate findings using multiple antibodies targeting different epitopes of C9orf78, or using complementary non-antibody-based methods.

How do I troubleshoot inconsistencies between published C9orf78 localization data?

When facing contradictory results regarding C9orf78 localization:

• Assess antibody validation status: Many discrepancies in the literature stem from inadequately validated antibodies. For example, studies on C9ORF72 (a different protein) used antibodies that later failed validation criteria, leading to contradictory localization reports .

• Consider fixation and permeabilization effects: Different methods can dramatically affect epitope accessibility and apparent protein localization. Systematically compare multiple fixation protocols to determine optimal conditions.

• Evaluate detection methods: Fluorescent tags versus antibody-based detection can yield different results. If possible, confirm localization using both approaches.

• Account for overexpression artifacts: Tagged overexpression can produce non-physiological localization. Compare endogenous protein localization with that of overexpressed proteins.

• Biological conditions: Cell type, cell cycle stage, and physiological state can influence protein localization. Standardize these variables when comparing results across studies.

What are the optimal conditions for using C9orf78 antibodies in Western blotting?

For optimal Western blot results with C9orf78 antibodies:

• Recommended dilution: For antibody 14496-1-AP, use at 1:500-1:3000 dilution as verified by manufacturer validation .

• Sample preparation: Prepare cell lysates from high-expressing cell lines like HeLa or HepG2 cells. Include appropriate positive controls.

• Protein loading: Load 20-50 μg of total protein per lane, adjusting based on expression level in your samples.

• Molecular weight confirmation: Verify that the detected band appears at the expected molecular weight of 34 kDa, which is the observed molecular weight for C9orf78 .

• Optimization steps: If signal is weak, consider:

  • Decreasing antibody dilution

  • Increasing protein loading

  • Extending primary antibody incubation (overnight at 4°C)

  • Using enhanced chemiluminescence detection systems

How should I optimize immunoprecipitation protocols for C9orf78?

For successful immunoprecipitation of C9orf78:

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation. For 14496-1-AP, use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Lysis buffer optimization: Use a buffer containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease inhibitors. Test different detergents if initial results are poor.

• Pre-clearing lysates: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody-bead conjugation: Pre-couple antibodies to protein A- or protein G-Sepharose as appropriate for the antibody isotype before adding cell lysate .

• Quantitative analysis: Consider using fluorescent secondary antibodies and imaging systems (like LI-COR) to quantitatively assess immunoprecipitation efficiency by analyzing both the immunoprecipitated fraction and the unbound supernatant .

• Validation: Confirm successful immunoprecipitation by immunoblotting the precipitated material with a different C9orf78 antibody that recognizes a separate epitope.

What are the best practices for immunohistochemistry with C9orf78 antibodies?

When performing immunohistochemistry with C9orf78 antibodies:

• Dilution optimization: For antibody 14496-1-AP, start with the recommended 1:50-1:500 dilution range and optimize for your specific tissue type .

• Antigen retrieval: Perform antigen retrieval with TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative depending on your tissue type .

• Controls: Include both positive and negative controls. Ideally, use tissue from C9orf78 knockout models as negative controls to confirm specificity .

• Detection system: For diaminobenzidine (DAB) labeling, optimize incubation times to achieve clear signal while minimizing background.

• Signal interpretation: Be cautious in interpreting staining patterns. Compare with published data and confirm specificity through multiple validation approaches, as non-specific binding can lead to misinterpretation of subcellular localization .

• Heat treatment: For some antibodies, treating sections at specific temperatures (e.g., 110°C) with appropriate pH buffers may be necessary for epitope unmasking and optimal staining .

How can I optimize immunofluorescence protocols for C9orf78 detection?

For successful immunofluorescence detection of C9orf78:

• Dilution range: For antibody 14496-1-AP, use within the recommended 1:200-1:800 dilution range for immunofluorescence applications .

• Cell preparation: HepG2 cells have been validated for immunofluorescence with this antibody . Fix cells with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100.

• Blocking optimization: Use 5% normal serum from the same species as your secondary antibody in PBS with 0.1% Triton X-100 for 1 hour at room temperature.

• Antibody incubation: Incubate with primary antibody overnight at 4°C, followed by fluorophore-conjugated secondary antibody (1:500-1:1000) for 1-2 hours at room temperature.

• Controls: Include both primary antibody omission controls and, ideally, C9orf78 knockout cell lines as negative controls to distinguish true signal from background autofluorescence .

• Counterstaining: Use DAPI or Hoechst for nuclear counterstaining to aid in localizing protein expression relative to cellular compartments.

How can I determine if my C9orf78 antibody recognizes all protein isoforms?

To assess isoform recognition:

• Sequence analysis: Compare the immunogen sequence used to generate the antibody against known C9orf78 isoforms. Antibody 14496-1-AP was generated using a fusion protein antigen (Ag5919) , so reviewing this sequence is critical.

• Recombinant protein testing: Test the antibody against recombinant proteins representing different isoforms. Commercial recombinant C9orf78 proteins, such as those with amino acids 1-289, can serve as positive controls .

• Cell-based validation: Express individual isoforms in cells lacking endogenous C9orf78 expression and test antibody recognition via Western blot.

• Mass spectrometry correlation: Correlate antibody detection with mass spectrometry data to confirm which specific isoforms are being detected in your experimental system.

• Isoform-specific knockdown: Use siRNA or shRNA targeting specific isoforms to determine which bands disappear on Western blot, helping identify which isoforms your antibody recognizes.

How can I establish a CRISPR/Cas9 knockout validation system for C9orf78 antibodies?

To implement a knockout validation system:

• Cell line selection: Choose cell lines with high endogenous C9orf78 expression, such as HeLa or HepG2 cells, which have documented expression .

• Guide RNA design: Design multiple guide RNAs targeting early exons of C9orf78 to ensure complete protein knockout.

• Knockout verification: Verify knockout through genomic sequencing of the targeted region and Western blot analysis using a previously validated antibody.

• Antibody testing pipeline:

  • Test new antibodies by immunoblot comparing parental and knockout cell lysates

  • Advance successful antibodies to immunoprecipitation testing

  • Further test promising candidates in immunofluorescence

  • Use the most robust antibodies for more complex applications like immunohistochemistry

• Functional validation: Consider complementation experiments by re-expressing C9orf78 in knockout cells to restore function and antibody reactivity.

How should I approach epitope mapping for C9orf78 antibodies?

For comprehensive epitope mapping:

• Peptide array analysis: Test antibody binding to overlapping peptides spanning the entire C9orf78 sequence to identify the specific binding region.

• Truncation constructs: Create a series of truncated C9orf78 constructs and test antibody binding to identify the minimal region required for recognition.

• Point mutations: Introduce systematic alanine substitutions within the identified binding region to pinpoint critical residues for antibody recognition.

• Cross-species comparison: If an antibody recognizes C9orf78 across species (human, mouse, rat) , compare sequences to identify conserved epitopes that likely contain the recognition site.

• Structural analysis: If structural data becomes available, correlate epitope mapping results with protein structure to assess epitope accessibility in the native protein.

How do I address non-specific binding issues with C9orf78 antibodies?

When encountering non-specific binding:

• Optimize blocking: Test different blocking agents (BSA, normal serum, commercial blockers) at various concentrations (3-5%) and incubation times (1-2 hours).

• Adjust antibody dilution: Titrate the antibody by testing a wider range of dilutions than recommended. For Western blot with 14496-1-AP, test dilutions from 1:250 to 1:5000 .

• Increase washing stringency: Add 0.1-0.5% Tween-20 to wash buffers and increase the number and duration of washing steps.

• Pre-adsorption: Pre-incubate the antibody with tissues or cell lysates from species not expressing the target protein to remove cross-reactive antibodies.

• Compare multiple antibodies: Test alternative antibodies targeting different epitopes of C9orf78 to determine if the non-specific binding is antibody-specific.

• Validate with knockout controls: Always compare results with C9orf78 knockout samples to definitively identify non-specific bands or staining patterns .

How can I quantitatively assess the affinity and specificity of C9orf78 antibodies?

For quantitative antibody assessment:

• Surface Plasmon Resonance (SPR): Determine binding kinetics (kon, koff) and equilibrium dissociation constant (KD) using purified C9orf78 protein and antibody.

• ELISA titration: Perform serial dilutions of antibody against a fixed amount of antigen to generate a binding curve and calculate apparent affinity.

• Competitive binding assays: Measure antibody displacement by free antigen to assess specificity and relative affinity.

• Flow cytometry: Quantify antibody binding to cells expressing C9orf78 versus knockout cells to determine specificity index and signal-to-noise ratio.

• Quantitative Western blot: Use fluorescent secondary antibodies and titration of both antibody and antigen to determine linear detection range and limit of detection.

• Cross-reactivity profiling: Test against a panel of related proteins to generate a comprehensive specificity profile.

How can I use C9orf78 antibodies for co-immunoprecipitation to identify interaction partners?

For successful co-immunoprecipitation studies:

• Antibody selection: Choose antibodies validated for immunoprecipitation, such as those recommended at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate .

• Crosslinking considerations: For transient interactions, consider using chemical crosslinkers like DSP or formaldehyde to stabilize protein complexes.

• Buffer optimization: Test different lysis conditions to preserve protein-protein interactions while maintaining effective extraction:

  • Low stringency: 0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5)

  • Medium stringency: 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5)

  • Add protease and phosphatase inhibitors to all buffers

• Negative controls: Include immunoprecipitation with non-specific IgG and, ideally, perform parallel experiments in C9orf78 knockout cells to identify non-specific interactors .

• Validation strategies: Confirm interactions by reverse co-immunoprecipitation using antibodies against the identified interaction partners.

• Mass spectrometry analysis: For unbiased interaction partner identification, analyze immunoprecipitates using LC-MS/MS and filter against results from control immunoprecipitations.

What considerations are important when using C9orf78 antibodies for super-resolution microscopy?

For super-resolution microscopy applications:

• Antibody specificity: Use only antibodies with exceptional specificity, as background fluorescence is particularly problematic in super-resolution techniques. Validate using knockout controls .

• Fluorophore selection: Choose bright, photostable fluorophores compatible with your super-resolution technique (STED, STORM, PALM).

• Secondary antibody considerations: For techniques requiring photoswitchable fluorophores (STORM/PALM), use high-quality secondary antibodies with appropriate dye-to-protein ratios.

• Sample preparation: Optimize fixation and permeabilization to preserve both protein localization and cellular ultrastructure:

  • Test both PFA and glutaraldehyde fixation at different concentrations

  • Compare different permeabilization agents (Triton X-100, saponin, digitonin)

• Antibody penetration: Ensure complete antibody penetration, particularly in tissue sections, by optimizing incubation times and conditions.

• Density considerations: For single-molecule localization techniques, optimize primary and secondary antibody concentrations to achieve appropriate labeling density.