C17orf78 Antibody

What is C17orf78 and what is its significance in research?

C17orf78 (Chromosome 17 Open Reading Frame 78) is an uncharacterized protein encoded by a gene located on the long arm cytogenetic band 17q12 of chromosome 17. The genomic sequence spans from position 37,375,985 to 37,392,708 on the forward strand, constituting 16,723 base pairs . The protein is highly expressed in the small intestine, particularly in the duodenum .

C17orf78 is significant in research as it represents one of the many proteins with potential but as-yet-undetermined functions in human biology. It is thought to play a role in immune system regulation, making it a promising target for studies on diseases such as cancer, autoimmune disorders, and inflammatory conditions . The characterization of such proteins advances our understanding of cellular pathways and potentially identifies new therapeutic targets.

What are the key characteristics of available C17orf78 antibodies?

Available C17orf78 antibodies, such as the C17orf78 Polyclonal Antibody (PACO39406), demonstrate the following characteristics:

• Host Species: Typically produced in rabbits

• Clonality: Available as polyclonal antibodies

• Isotype: Generally IgG

• Species Reactivity: Human-specific

• Validated Applications: ELISA, IHC (immunohistochemistry), and IF (immunofluorescence)

• Form: Usually supplied in liquid form

These antibodies are designed to bind specifically to the C17orf78 protein, allowing researchers to detect and analyze its expression in various cell types and tissues.

What applications are C17orf78 antibodies validated for?

C17orf78 antibodies have been validated for multiple research applications, with specific recommended dilutions:

These applications have been validated with specific tissues and cell lines. For example, immunohistochemistry has been validated using paraffin-embedded human spleen tissue, while immunofluorescence analysis has been performed on A549 cells using Alexa Fluor 488-conjugated secondary antibodies .

How can researchers validate the specificity of C17orf78 antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For C17orf78 antibodies, researchers should implement a multi-faceted validation approach:

• Knockout/knockdown controls: Generate CRISPR-Cas9 knockout cell lines or siRNA knockdown of C17orf78 to compare with wild-type expression patterns. This approach follows established practices for protein characterization, similar to methods used for validating other proteins like tyrosine-protein kinase SYK .

• Overexpression validation: Use recombinant C17orf78 expression vectors, such as the available bacterial expression vectors with His tags , to create overexpression models for antibody testing.

• Western blot analysis: Perform detailed molecular weight assessment. The primary C17orf78 isoform is expected to appear at approximately 30.55 kDa with an isoelectric point of 9.62 .

• Cross-reactivity testing: Test the antibody against related proteins to ensure specificity, particularly against proteins expressed in similar tissues.

• Absorption controls: Pre-absorb the antibody with purified C17orf78 protein before immunostaining to confirm binding specificity.

What are the methodological considerations for detecting the different isoforms of C17orf78?

C17orf78 has two known splice variant isoforms:

• Isoform 1 (C17orf78-204): 275 amino acids, includes all 7 exons, considered the principal protein

• Isoform 2 (C17orf78-203): 159 amino acids, constituted from 5 exon regions (1st, 2nd, 3rd, 6th, and 7th exons)

To effectively distinguish between these isoforms, researchers should:

• Select antibodies with appropriate epitope recognition: Choose antibodies targeting regions that differ between isoforms, particularly those recognizing the 4th and 5th exons present only in isoform 1.

• Use high-resolution gel electrophoresis: Employ gradient gels that can clearly separate the 30.55 kDa (isoform 1) from the smaller isoform 2.

• Implement isoform-specific PCR: Design primers that selectively amplify each isoform to correlate protein detection with transcript expression.

• Consider mass spectrometry: For unambiguous identification, use MS-based proteomics to identify isoform-specific peptides.

How can researchers optimize immunofluorescence protocols for C17orf78 detection?

Optimizing immunofluorescence protocols for C17orf78 requires careful attention to several parameters:

• Fixation method: Compare paraformaldehyde (4%) with methanol fixation to determine which better preserves C17orf78 epitopes.

• Permeabilization optimization: Test various detergents (0.1-0.5% Triton X-100, 0.1% Saponin) to optimize access to the epitope while preserving cellular structures.

• Antibody dilution: Begin with the recommended dilution range (1:50-1:200) and perform titration experiments to determine optimal signal-to-noise ratio.

• Incubation conditions: Compare overnight incubation at 4°C with shorter incubations at room temperature.

• Secondary antibody selection: For optimal results, use Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L), which has been validated for C17orf78 detection .

• Mounting media optimization: Use anti-fade mounting media containing DAPI for nuclear counterstaining.

What sample preparation techniques are recommended for C17orf78 antibody applications?

For optimal results with C17orf78 antibodies, sample preparation techniques should be tailored to the application:

For Western Blot:

• Use RIPA buffer supplemented with protease inhibitors for protein extraction

• Sonicate samples briefly to fragment genomic DNA

• Centrifuge at 12,000g for 15 minutes at 4°C to remove debris

• Heat proteins in Laemmli buffer at 95°C for 5 minutes before loading

• Load 20-40 μg of total protein per lane

For Immunohistochemistry:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process tissues through graded alcohols and xylene

• Embed in paraffin and section at 4-6 μm thickness

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Use protein blocking solution to reduce non-specific binding

For Immunofluorescence:

• Culture cells on glass coverslips to 70-80% confluence

• Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 5% normal goat serum in PBS for 1 hour

• Incubate with primary antibody at recommended dilutions (1:50-1:200)

How should researchers troubleshoot weak or non-specific signals when using C17orf78 antibodies?

When encountering weak or non-specific signals with C17orf78 antibodies, researchers should implement a systematic troubleshooting approach:

For Weak Signals:

• Increase antibody concentration: Adjust the dilution factor toward the more concentrated end of the recommended range (ELISA: 1:2000, IHC: 1:20, IF: 1:50)

• Extend incubation time: Increase primary antibody incubation from overnight to 48 hours at 4°C

• Enhance epitope retrieval: Test different antigen retrieval methods (citrate buffer, EDTA buffer, enzymatic retrieval)

• Use signal amplification: Implement tyramide signal amplification or biotin-streptavidin systems

• Optimize protein loading: Increase the amount of total protein loaded per well for Western blots

For Non-specific Signals:

• Increase blocking: Use 5% BSA or 10% normal serum from the secondary antibody host species

• Add detergent: Include 0.1-0.3% Tween-20 in washing and antibody dilution buffers

• Pre-absorb antibody: Incubate with irrelevant proteins to reduce non-specific binding

• Reduce primary antibody concentration: Dilute further if background is high

• Optimize washing: Increase the number and duration of wash steps

What are the recommended storage conditions for maintaining C17orf78 antibody activity?

To maintain optimal activity of C17orf78 antibodies, observe these storage recommendations:

• Long-term storage: Store antibodies at -20°C, divided into small aliquots to avoid repeated freeze-thaw cycles

• Working dilutions: Store diluted antibodies at 4°C for up to one week

• Storage buffer: The antibody is stable in its provided buffer (50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative)

• Avoid contamination: Use sterile technique when handling antibodies

• Monitor stability: Record lot number and date of first use to track potential degradation over time

How should researchers interpret variable C17orf78 expression patterns across different tissues?

When analyzing C17orf78 expression patterns across different tissues, researchers should:

• Establish baseline expression: First document expression in tissues known to have high C17orf78 levels, such as the small intestine, particularly the duodenum .

• Perform quantitative analysis: Use image analysis software to quantify staining intensity across different tissues, normalizing to appropriate housekeeping proteins.

• Consider tissue context: Interpret expression patterns in relation to the tissue's function. For example, high expression in intestinal tissues may suggest a role in digestive or immune functions.

• Correlate with transcript data: Compare protein expression with available RNA-seq or qPCR data to validate findings.

• Evaluate cellular localization: Document subcellular localization patterns (nuclear, cytoplasmic, membrane-associated) as these may provide functional clues.

• Analyze isoform distribution: Consider whether different tissues preferentially express specific isoforms, which may indicate tissue-specific functions.

What bioinformatic approaches can help predict potential functions of C17orf78?

Several bioinformatic approaches can help researchers predict potential functions of uncharacterized proteins like C17orf78:

• Sequence homology analysis: Identify conserved domains or motifs that may suggest functional roles.

• Protein-protein interaction prediction: Use tools like STRING or BioGRID to predict interaction partners that may provide functional context.

• Co-expression analysis: Identify genes with similar expression patterns across tissues and conditions, as co-expressed genes often participate in related biological processes.

• Structural prediction: Use tools like AlphaFold to predict protein structure, which can provide insights into potential functions.

• Pathway enrichment analysis: Analyze the functional enrichment of predicted interaction partners to identify potential pathways involving C17orf78.

• Evolutionary conservation analysis: Examine conservation across species to identify functionally important regions.

How can researchers design experiments to investigate the potential immune regulatory function of C17orf78?

Based on the suggested role of C17orf78 in immune regulation , researchers can design the following experimental approaches:

• Cell type-specific expression profiling: Quantify C17orf78 expression across different immune cell populations using flow cytometry and immunofluorescence.

• Stimulus-response experiments: Expose immune cells to various stimuli (cytokines, PAMPs, DAMPs) and monitor changes in C17orf78 expression.

• Loss-of-function studies: Use siRNA or CRISPR-Cas9 to knock down or knock out C17orf78 in immune cells and assess the impact on:

  • Cytokine production

  • Cell activation markers

  • Proliferation rates

  • Phagocytic capacity

  • Migration ability

• Gain-of-function studies: Overexpress C17orf78 using expression vectors and analyze the resulting phenotypes.

• In vivo models: Generate conditional knockout mouse models to study tissue-specific effects of C17orf78 deficiency on immune responses.

• Disease model analysis: Examine C17orf78 expression in samples from patients with inflammatory or autoimmune diseases to identify potential correlations with disease progression.

What are promising approaches for elucidating the functional role of C17orf78?

Several strategies can advance our understanding of C17orf78 function:

• Proteomics-based interactome mapping: Perform immunoprecipitation followed by mass spectrometry to identify C17orf78 binding partners, potentially revealing functional pathways.

• CRISPR screens: Conduct genome-wide CRISPR screens in cells expressing C17orf78 to identify genetic interactions and potential functional pathways.

• Single-cell analysis: Use single-cell RNA-seq and proteomics to characterize cell types expressing C17orf78 and correlate expression with cellular states.

• Structural biology: Determine the three-dimensional structure of C17orf78 to provide insights into potential molecular functions.

• Evolutionary analysis: Compare C17orf78 orthologs across species to identify conserved domains likely to be functionally important.

• Transgenic animal models: Generate reporter mice to visualize C17orf78 expression patterns in vivo during development and in response to various stimuli.

How might researchers integrate C17orf78 antibody data with other omics approaches?

Integrating antibody-based detection with multi-omics approaches offers a comprehensive understanding of C17orf78:

• Antibody-verified proteomics: Use antibody-enriched samples for targeted proteomics to increase sensitivity for low-abundance C17orf78.

• Spatial transcriptomics with protein validation: Correlate spatial transcriptomics data with immunohistochemistry to verify transcript-protein relationships in tissue context.

• ChIP-seq validation: Use C17orf78 antibodies for ChIP-seq if the protein is suspected to interact with chromatin, followed by validation with immunofluorescence co-localization studies.

• PTM mapping: Combine antibodies recognizing specific post-translational modifications with mass spectrometry to characterize C17orf78 regulation.

• Integrated network analysis: Combine antibody-based interaction data with transcriptomic networks to build comprehensive functional models of C17orf78 in cellular pathways.