C7orf31 Antibody

What is C7orf31 and why are antibodies against it important for research?

C7orf31 is an abbreviation for "Chromosome 7 Open Reading Frame 31," which represents a protein-coding gene located on chromosome 7. The protein is largely uncharacterized, with alternative names including "uncharacterized protein C7orf31" and "chromosome 2 C7orf31 homolog (C2H7orf31)" . Antibodies against C7orf31 are critical research tools that enable detection, quantification, and localization of this protein in various experimental contexts. While the function of C7orf31 remains incompletely understood, antibodies provide essential means to investigate its expression patterns, cellular localization, and potential interactions with other biomolecules.

What applications are C7orf31 antibodies commonly used for in research?

C7orf31 antibodies have demonstrated utility across multiple research applications, primarily:

• Western Blotting (WB): For detecting and quantifying C7orf31 protein in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative determination of C7orf31 concentrations

• Immunofluorescence (IF): For visualizing cellular localization patterns

The specific applications vary by antibody, with some validated for multiple techniques while others are optimized for singular applications . When designing experiments, researchers should select antibodies specifically validated for their intended application, as performance can vary significantly across different techniques.

How do I select the appropriate C7orf31 antibody for my research?

Selection of an appropriate C7orf31 antibody requires consideration of several key factors:

• Species reactivity: Determine which species your samples derive from and ensure the antibody has demonstrated reactivity to that species. Available C7orf31 antibodies show varying reactivity profiles including human-specific antibodies and those with cross-reactivity to mouse, rat, hamster, monkey, and rabbit samples .

• Application validation: Verify that the antibody has been validated for your specific application (WB, ELISA, IF). Some antibodies perform well in multiple applications while others are application-specific .

• Validation quality: Consider the extent of validation data available. Higher-quality antibodies typically demonstrate validation across multiple cell lines or tissue types with appropriate controls.

• Format compatibility: Ensure the antibody format (monoclonal vs. polyclonal) is suitable for your experimental design.

Following similar principles to established antibody validation pipelines, researchers should ideally test multiple antibodies against C7orf31 to identify those with highest specificity and sensitivity for their experimental system .

What controls should I include when using C7orf31 antibodies?

Proper experimental controls are essential when using C7orf31 antibodies to ensure result validity:

Positive Controls:

• Cell lines known to express C7orf31 (check expression databases)

• Recombinant C7orf31 protein (if available)

• Overexpression lysates from cells transfected with C7orf31 expression vector

Negative Controls:

• CRISPR/Cas9 knockout cell lines lacking C7orf31 expression

• Samples treated with siRNA targeting C7orf31

• Cell lines with naturally low or no expression of C7orf31

Technical Controls:

• Primary antibody omission control

• Isotype control antibody

• Blocking peptide competition (if available)

The gold standard negative control for antibody validation is the use of knockout cell lines, which should show complete absence of signal when probed with a specific antibody . This approach has been successfully implemented for validating other ORF antibodies and can be adapted for C7orf31 .

How can I validate a commercial C7orf31 antibody using CRISPR/Cas9 knockout approaches?

Validating C7orf31 antibodies using CRISPR/Cas9 knockout approaches requires a systematic workflow:

• Cell line selection: Using a proteomics database like PaxDB (https://pax-db.org/), identify cell lines with relatively high C7orf31 expression that are amenable to CRISPR/Cas9 editing .

• CRISPR/Cas9 knockout generation:

  • Design guide RNAs targeting early exons of C7orf31

  • Transfect target cells with Cas9 and guide RNAs

  • Screen and isolate clonal populations

  • Verify genetic modification by sequencing

• Antibody screening:

  • Perform immunoblot comparing parental and knockout cell lines

  • Valid antibodies will show bands of expected molecular weight in parental cells that are absent in knockout cells

  • Test multiple antibodies in parallel to identify those with highest specificity

• Cross-application validation:

  • For antibodies passing the immunoblot screen, expand testing to other applications like immunoprecipitation and immunofluorescence

  • Compare signal patterns between parental and knockout lines across applications

This approach mirrors successful validation strategies employed for other ORF proteins like C9ORF72, where knockout controls revealed that many commercially available antibodies lacked specificity despite their widespread use in published literature .

What are the methodological considerations for using C7orf31 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) using C7orf31 antibodies requires careful optimization:

Antibody Selection:

• Choose antibodies validated specifically for immunoprecipitation

• Antibodies that perform well in immunoblot may not necessarily function in IP applications, as observed with other ORF antibodies

• Pre-screen multiple antibodies by performing test IPs followed by immunoblot detection

Experimental Protocol Optimization:

• Lysis buffer selection: Use buffers that preserve protein-protein interactions while efficiently extracting C7orf31 (typically RIPA or NP-40-based buffers)

• Pre-clearing strategy: Implement sample pre-clearing with protein A/G beads to reduce non-specific binding

• Antibody coupling: Pre-couple antibodies to protein A/G beads for optimal capture efficiency

• Washing stringency: Balance between removing non-specific interactions and preserving genuine interactions

• Elution conditions: Optimize to ensure complete recovery of precipitated complexes

Controls and Validation:

• Include IgG control immunoprecipitations

• Compare IPs from parental versus C7orf31-knockout cells

• Validate interactions through reciprocal IPs when possible

• Consider mass spectrometry analysis of immunoprecipitates to identify C7orf31-interacting proteins comprehensively

Following approaches similar to those used for C9ORF72, quantification of immunoprecipitation efficiency can be performed using fluorescent secondary antibodies and imaging systems like LI-COR Odyssey to determine the percentage of target protein depleted from the supernatant .

How should immunofluorescence protocols be optimized for C7orf31 detection in different cell types?

Optimizing immunofluorescence protocols for C7orf31 requires systematic adjustment of multiple parameters:

Sample Preparation Variables:

• Fixation method: Compare paraformaldehyde (4%) versus methanol fixation

• Permeabilization: Test different detergents (0.1-0.5% Triton X-100, 0.1% Saponin)

• Blocking conditions: Optimize blocking buffer composition (BSA percentage, serum type)

• Epitope retrieval: Evaluate necessity of antigen retrieval methods

Antibody Parameters:

• Dilution optimization: Test serial dilutions to maximize signal-to-noise ratio

• Incubation conditions: Compare room temperature versus 4°C incubation, varying durations

• Secondary antibody selection: Choose fluorophores compatible with available microscopy setup

Validation Approach:

• Include C7orf31 knockout cells as negative controls

• Perform peptide competition assays if blocking peptides are available

• Consider co-localization with known organelle markers to characterize subcellular distribution

Cell Type Considerations:

• Different cell types may require distinct optimization conditions

• Expression levels may vary significantly across cell types, requiring adjustment of antibody concentrations

• Compare endogenous staining patterns with GFP-tagged C7orf31 expression when possible

The subcellular localization pattern observed should be consistent across multiple validated antibodies to confirm specificity, as has been demonstrated in validation studies for other ORF proteins .

What approaches can resolve contradictory results obtained with different C7orf31 antibodies?

Contradictory results from different C7orf31 antibodies represent a common challenge in research. A systematic troubleshooting approach includes:

Comprehensive Antibody Validation:

• Parallel antibody testing: Screen all available antibodies simultaneously using CRISPR/Cas9 knockout controls

• Epitope mapping: Determine the exact epitopes recognized by each antibody

• Isoform specificity: Assess whether discrepancies arise from differential detection of protein isoforms

Technical Resolution Strategies:

• Orthogonal method confirmation: Verify results using non-antibody-based methods (e.g., mass spectrometry)

• Tagged protein expression: Compare antibody results with detection of epitope-tagged C7orf31

• Genetic manipulation: Use RNAi and overexpression to confirm signal specificity

Data Integration Framework:

This approach mirrors successful strategies used to resolve contradictory C9ORF72 localization reports, where comprehensive antibody validation revealed that many previously reported localizations were based on non-specific antibody signals .

What considerations are important when using C7orf31 antibodies for immunohistochemistry in tissue sections?

Using C7orf31 antibodies for immunohistochemistry (IHC) requires addressing tissue-specific challenges:

Tissue Processing Parameters:

• Fixation method: Compare performance in formalin-fixed versus frozen tissues

• Antigen retrieval: Systematically test different retrieval methods:

  • Heat-induced epitope retrieval at varying temperatures (95-120°C)

  • pH optimization (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Enzymatic retrieval approaches

Antibody Optimization:

• Titration: Determine optimal antibody concentration for each tissue type

• Incubation conditions: Test various durations (overnight vs. shorter incubations)

• Detection system: Compare sensitivity of DAB, fluorescence, or amplification systems

Controls Implementation:

• Tissue from knockout animals: Essential negative control if available

• Peptide competition: Pre-absorb antibody with immunizing peptide

• Regional variation analysis: Compare staining patterns in regions with expected differential expression

Result Interpretation:

• Document and quantify cellular and subcellular distribution patterns

• Distinguish between specific signal and common artifacts

• Correlate IHC results with other expression data (RNA-seq, proteomics)

Experience with C9ORF72 antibodies demonstrates that antibodies performing well in immunoblot may not necessarily work for IHC and vice versa. For example, GTX634482 antibody showed specific staining in brain sections while ab221137, despite working well for immunoblot, showed no signal in IHC analysis .

What are the current limitations in C7orf31 antibody research and future directions?

Current limitations in C7orf31 antibody research largely mirror challenges faced with other uncharacterized ORF proteins:

Current Limitations:

• Incomplete validation of many commercially available antibodies

• Limited knowledge of protein function impeding functional validation

• Potential variability in protein expression across different tissues and conditions

• Possibility of multiple isoforms with distinct epitope availability

Future Research Directions:

• Comprehensive antibody validation:

  • Systematic validation using CRISPR/Cas9 knockout controls

  • Cross-application testing for each antibody

  • Development of renewable antibody resources (recombinant antibodies)

• Functional characterization:

  • Integration of antibody-based detection with functional assays

  • Investigation of C7orf31 in various cellular processes

  • Identification of regulatory mechanisms controlling expression

• Technical advances:

  • Development of proximity labeling approaches for interaction studies

  • Integration with emerging proteomic technologies

  • Creation of knockin cell lines with endogenous tags for validation

• Collaborative approaches:

  • Establishment of community standards for antibody validation

  • Creation of shared resources for validated reagents

  • Integration of data across multiple research groups