C8orf34 Antibody

What is C8orf34 and why is it relevant to antibody-based research?

C8orf34 (chromosome 8 open reading frame 34) is a protein encoded by the C8orf34 gene in humans. It is also known as vestibule-1 or VEST-1. C8orf34 is primarily localized to the nucleus and nucleoli, suggesting its potential role in gene expression regulation and cell cycle processes .

The protein lacks a signal peptide that would allow it to sort outside the nuclear membrane or to other organelles. Analysis via PSORT II has shown that C8orf34 is localized to the nucleus with 94.1% reliability . This nuclear localization indicates that C8orf34 likely functions in the expression and regulation of genes or in the maintenance and protection of genetic material.

C8orf34 is widely expressed across various tissues, including kidney, stomach, thymus, pituitary gland, ear, and brain. Within the brain, it shows expression in specific regions such as the dentate gyrus, epithalamus, and medulla . This diverse expression pattern makes C8orf34 antibodies valuable tools for studying tissue-specific functions of this protein.

What are the available types of C8orf34 antibodies and their applications?

Several types of C8orf34 antibodies are available for research applications. One common type is polyclonal antibodies, such as biotin-conjugated polyclonal antibodies suitable for ELISA applications . These antibodies are typically generated by immunizing rabbits with recombinant C8orf34 protein fragments.

The properties of a typical C8orf34 polyclonal antibody include:

These antibodies can be employed in various research applications including ELISA, Western blotting, immunohistochemistry, and potentially flow cytometry, though specific validation for each application is necessary .

How can I validate the specificity of a C8orf34 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For C8orf34 antibodies, consider implementing these methodological approaches:

Positive and Negative Control Lysates

Use tissue or cell lysates with known C8orf34 expression levels. Based on expression data, kidney, stomach, thymus, and certain brain regions would serve as positive controls, while tissues with minimal expression could serve as negative controls .

Knockdown/Knockout Validation

Generate C8orf34 knockdown cell lines using siRNA or CRISPR-Cas9 technology similar to the approach used for C8orf34-as1 in lung cancer studies . Compare antibody signal between wild-type and knockdown cells—a specific antibody will show reduced signal in knockdown samples.

Peptide Competition Assay

Pre-incubate the antibody with excess purified C8orf34 protein or immunogenic peptide before application. Specific binding will be blocked, resulting in signal reduction or elimination.

Multiple Antibody Validation

Compare results from different antibodies targeting distinct epitopes of C8orf34. Consistent results across different antibodies increase confidence in specificity.

Mass Spectrometry Confirmation

Following immunoprecipitation with the C8orf34 antibody, verify the precipitated protein using mass spectrometry techniques similar to those described for intact protein verification .

What is the optimal protocol for Western blotting using C8orf34 antibodies?

When conducting Western blot analysis with C8orf34 antibodies, follow this research-optimized protocol:

Sample Preparation

• Extract total protein from tissues or cells using RIPA buffer supplemented with protease inhibitors.

• Quantify protein concentration using Bradford or BCA assay.

• Prepare 20-50 μg of total protein per lane with loading buffer containing DTT or β-mercaptoethanol.

• Heat samples at 95°C for 5 minutes for complete denaturation.

Gel Electrophoresis and Transfer

• Separate proteins on 10-12% SDS-PAGE gels (C8orf34 has a molecular weight that would be well resolved in this range).

• Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes in cold transfer buffer.

Antibody Incubation

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Incubate with primary C8orf34 antibody at 1:500-1:1000 dilution in blocking solution overnight at 4°C.

• Wash membrane 3 times with TBST, 5 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 dilution for 1 hour at room temperature.

• Wash membrane 3 times with TBST, 5 minutes each.

Detection and Analysis

• Apply ECL substrate and capture signal using digital imaging system.

• Include appropriate loading controls (GAPDH, β-actin) for normalization.

• Perform densitometric analysis to quantify relative expression levels.

This protocol can be adapted for detecting C8orf34 in nuclear fraction preparations, which would be particularly relevant given its nuclear localization .

How can I utilize C8orf34 antibodies in studying nuclear protein complexes?

Given C8orf34's nuclear and nucleolar localization , antibodies against this protein are valuable tools for studying nuclear protein complexes:

Co-Immunoprecipitation (Co-IP) Protocol

• Prepare nuclear extracts from cells of interest using a nuclear protein extraction kit.

• Pre-clear nuclear lysate with Protein A/G beads for 1 hour at 4°C.

• Incubate pre-cleared lysate with C8orf34 antibody (2-5 μg) overnight at 4°C with gentle rotation.

• Add Protein A/G beads and incubate for 2-3 hours at 4°C.

• Wash beads 4-5 times with low-salt wash buffer.

• Elute bound proteins with SDS sample buffer and analyze by Western blot or mass spectrometry.

Chromatin Immunoprecipitation (ChIP)

Since C8orf34 may be involved in gene regulation , ChIP can identify DNA sequences it might associate with:

• Cross-link protein-DNA complexes with 1% formaldehyde for 10 minutes.

• Lyse cells and sonicate to shear chromatin (200-500 bp fragments).

• Immunoprecipitate with C8orf34 antibody overnight at 4°C.

• Wash, elute, reverse cross-links, and purify DNA.

• Analyze by qPCR, focusing on promoter regions of genes regulated by transcription factors known to interact with C8orf34, such as OCT1, STAT3, HSF1, or MZF1 .

Proximity Ligation Assay (PLA)

This technique can visualize protein-protein interactions in situ:

• Fix cells on coverslips with 4% paraformaldehyde.

• Permeabilize with 0.2% Triton X-100.

• Block and incubate with C8orf34 antibody and antibody against potential interacting protein.

• Follow PLA protocol with appropriate PLA probes.

• Analyze using confocal microscopy, focusing on nuclear signals.

What role does C8orf34 play in cancer research and how can antibodies help investigate this?

C8orf34 and its antisense transcript C8orf34-as1 have been implicated in cancer research, particularly in lung adenocarcinoma (LUAD). C8orf34-as1 has been identified as part of a competing endogenous RNA (ceRNA) regulatory axis that influences cancer stemness and cell invasion in LUAD .

C8orf34-as1/miR-671/MFAP4 Regulatory Axis

Research has shown that C8orf34-as1 acts as a ceRNA by competitively sponging miR-671-5p to regulate MFAP4 expression. In LUAD tissues, C8orf34-as1 and MFAP4 expression is significantly lower than in normal lung tissues, while miR-671 expression is higher .

Correlation analysis revealed:

• MFAP4 positively correlates with C8orf34-as1 expression (r = 0.2125, p < 0.0001)

• MFAP4 negatively correlates with miR-671-5p expression (r = -0.3113, p < 0.0001)

• C8orf34-as1 negatively correlates with miR-671-5p levels (r = -0.3244, p < 0.0001)

Antibody-Based Research Applications

• Immunohistochemistry (IHC): C8orf34 antibodies can be used to compare protein expression between normal and cancerous tissues, potentially serving as diagnostic or prognostic markers.

• Functional Studies: Researchers can use C8orf34 antibodies in combination with techniques like RNA interference to investigate how modulating C8orf34 affects cancer cell phenotypes.

• Therapeutic Potential: Given the development of antibody-based therapeutics for cancer, researchers might explore C8orf34 antibodies as potential targeting agents, similar to approaches used for other cancer-associated proteins .

RT-qPCR Protocol for C8orf34 mRNA Analysis

While not directly antibody-based, RT-qPCR provides valuable complementary data to antibody-based protein detection:

• Extract total RNA using TRIzol reagent.

• Synthesize cDNA using a TaqMan real-time PCR kit.

• Perform RT-PCR using a SYBR Green qPCR Kit.

• Use appropriate primers for C8orf34 (similar to those designed for C8orf34-as1) :

  Primer	Sequence
  Forward	5′-CAGGGGACCGATCTTGTTCT-3′
  Reverse	5′-AGTGCTCAGTCTTCACCTT-3′

• Use GAPDH as an internal control with primers:

  Primer	Sequence
  Forward	5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′
  Reverse	5′-CATGTGGGCCATGAGGTCCACCAC-3′

• Calculate relative expression using the 2^(-ΔΔCT) method.

Immunohistochemistry (IHC) Protocol

• Prepare formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm).

• Deparaffinize and rehydrate sections.

• Perform antigen retrieval (citrate buffer pH 6.0, pressure cooker method recommended).

• Block endogenous peroxidase with 3% H₂O₂.

• Block non-specific binding with 5% normal serum.

• Incubate with primary C8orf34 antibody overnight at 4°C (optimize dilution, typically 1:100-1:500).

• Wash and apply appropriate secondary antibody.

• Develop with DAB and counterstain with hematoxylin.

• Score staining intensity and percentage of positive cells.

Given C8orf34's nuclear localization , pay particular attention to nuclear staining patterns.

How do transcription factors influence C8orf34 expression, and how might this affect antibody-based studies?

Several transcription factors regulate C8orf34 gene expression, many related to cell cycle regulation and longevity. This regulatory network suggests C8orf34 may function in these processes .

Implications for Antibody-Based Research:

• Cell Stress Response Studies: When using C8orf34 antibodies in stress response experiments, consider that HSF1 activation may alter C8orf34 expression, potentially confounding results.

• Cell Cycle Analysis: For cell cycle studies, synchronize cells and use C8orf34 antibodies in combination with cell cycle markers to track expression changes throughout different phases.

• Cell Type Considerations: Due to MZF1's role in hematopoietic cell differentiation, expression of C8orf34 may vary significantly in these cell types, requiring careful interpretation of antibody-based detection.

• Experimental Design: When manipulating pathways involving these transcription factors, monitor C8orf34 expression changes using antibody-based methods like Western blotting or immunofluorescence.

• STAT3 Pathway Interactions: In inflammatory or cancer studies involving STAT3 signaling, include C8orf34 antibody detection to explore potential mechanistic connections.

Common Issues and Solutions in Western Blotting

• Weak Signal

  • Increase antibody concentration (try 1:250 instead of 1:500)

  • Extend primary antibody incubation time to overnight at 4°C

  • Use more sensitive detection system (e.g., enhanced chemiluminescence)

  • Increase protein loading (up to 50-75 μg per lane)

  • Consider nuclear extraction protocols to enrich for C8orf34 given its nuclear localization

• Multiple Bands/Nonspecific Binding

  • Increase blocking time or blocking agent concentration (5-10% blocking solution)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Perform peptide competition assay to identify specific bands

  • Use freshly prepared samples to minimize degradation

  • Increase wash duration and number of washes

  • Consider using monoclonal antibodies for higher specificity

• High Background

  • Reduce antibody concentration

  • Increase blocking agent concentration or blocking time

  • Use alternative blocking agents (switch between milk and BSA)

  • Include 0.05% sodium azide in antibody dilution buffer to prevent bacterial growth

  • Ensure clean preparation of transfer membrane

Troubleshooting in Immunocytochemistry/Immunohistochemistry

• No Signal or Weak Signal

  • Optimize antigen retrieval method (try citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic retrieval)

  • Increase antibody concentration

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use tyramide signal amplification system

  • Verify tissue fixation protocol (overfixation may mask epitopes)

• High Background/Nonspecific Staining

  • Include additional blocking steps (e.g., avidin/biotin blocking for biotin-conjugated antibodies)

  • Reduce primary antibody concentration

  • Include 0.1-0.3% Triton X-100 in wash buffer

  • Increase wash steps duration and frequency

  • Pre-absorb antibody with tissue powder

What considerations are important when designing a C8orf34 ELISA assay?

ELISA is a key application mentioned for C8orf34 antibodies . Here's a comprehensive protocol and troubleshooting guide:

Sandwich ELISA Protocol for C8orf34

• Plate Preparation

  • Coat 96-well Maxisorp plates with capture antibody (1-10 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C

  • Alternatively, use NeutrAvidin (10 μg/ml) coating for biotinylated antibodies

• Blocking

  • Block with 2% BSA in PBS for 1-2 hours at room temperature

• Sample and Standard Addition

  • Prepare a standard curve using recombinant C8orf34 protein

  • Dilute samples appropriately in ELISA buffer (PBS, pH 7.4, 0.05% Tween-20, 0.2% BSA)

  • Incubate for 1-2 hours at room temperature

• Detection Antibody

  • Add biotinylated detection antibody at optimized concentration

  • Incubate for 1 hour at room temperature

• Signal Development

  • Add streptavidin-HRP (1:5000-1:10000) and incubate for 30-60 minutes

  • Wash and add TMB substrate

  • Stop reaction with 2N H₂SO₄ after appropriate color development

  • Read absorbance at 450nm with 570nm reference

Optimization Considerations

• Antibody Pair Selection

  • Use antibodies recognizing different epitopes for capture and detection

  • Test multiple combinations to identify the most sensitive and specific pair

  • Consider using biotinylated antibodies for detection to enhance sensitivity

• Sample Preparation

  • For cell/tissue lysates, use RIPA buffer with protease inhibitors

  • Centrifuge lysates at high speed to remove cellular debris

  • Determine optimal sample dilution through preliminary titration experiments

• Assay Validation

  • Determine lower limit of detection and quantification

  • Assess intra-assay and inter-assay variability (<15% CV is desirable)

  • Perform spike-and-recovery experiments to validate accuracy

  • Test linearity of dilution to confirm proportional detection across concentration range

How can C8orf34 antibodies be used to investigate the competing endogenous RNA regulatory networks?

The search results reveal an important role for C8orf34-as1 in a competing endogenous RNA (ceRNA) regulatory axis in lung adenocarcinoma . While this focuses on the antisense transcript rather than C8orf34 protein itself, antibodies against C8orf34 can be valuable in investigating related regulatory networks:

Research Approach for ceRNA Network Investigation

• Dual Analysis Strategy

  • Combine C8orf34 antibody-based protein detection with RNA analysis of C8orf34-as1

  • Investigate whether C8orf34 protein levels correlate with C8orf34-as1 expression patterns

• Integrated RNA-Protein Analysis Protocol

  • Extract protein and RNA from the same samples

  • Perform RT-qPCR for C8orf34-as1 and miR-671-5p expression using protocols similar to those described

  • Use Western blotting with C8orf34 antibodies to detect protein levels

  • Analyze correlations between C8orf34 protein levels and C8orf34-as1/miR-671-5p expression

• Functional Studies

  • Overexpress or knockdown C8orf34-as1 and measure changes in C8orf34 protein levels

  • Use C8orf34 antibodies in immunoprecipitation studies to identify potential protein interaction partners that might be affected by the ceRNA network

  • Perform dual-luciferase reporter assays similar to those used for validating the C8orf34-as1/miR-671-5p interaction

• Subcellular Localization Analysis

  • Use C8orf34 antibodies in immunofluorescence microscopy to determine subcellular localization

  • Compare localization patterns under conditions where the ceRNA network is perturbed

  • Analyze whether C8orf34 co-localizes with components of the miRNA processing machinery

How does C8orf34's role differ between normal and disease contexts?

Based on the search results, C8orf34 and its antisense transcript C8orf34-as1 show differential expression between normal and cancer tissues, particularly in lung adenocarcinoma . Here's how researchers can investigate these differences:

Comparative Expression Analysis

• Tissue Microarray (TMA) Analysis

  • Use C8orf34 antibodies to perform IHC on TMAs containing normal and cancerous tissues

  • Quantify staining intensity and distribution, particularly focusing on nuclear localization

  • Correlate expression patterns with clinical parameters and patient outcomes

• Cell Line Panel Analysis

  • Compare C8orf34 protein levels across normal and cancer cell lines using Western blotting

  • Include cells from different cancer stages to assess potential correlation with disease progression

  • Combine with RT-qPCR analysis of C8orf34-as1 to examine protein-RNA relationships

Functional Investigation Protocol

• Manipulation of Expression Levels

  • Overexpress C8orf34 in cancer cell lines with low endogenous expression

  • Knockdown C8orf34 in normal cells or cancer cells with high expression

  • Assess effects on:

    • Cell proliferation (using assays like CellTiter-Glo )

    • Cell invasion (using Transwell assays as described for MFAP4 studies )

    • Cancer stemness (using tumor sphere formation assays )

• Pathway Analysis

  • Perform Western blotting with antibodies against C8orf34 and stemness markers (SOX2, OCT4, NANOG) after manipulating C8orf34 expression

  • Investigate interactions with transcription factors known to regulate C8orf34 (OCT1, STAT3, HSF1, MZF1)

  • Use phospho-specific antibodies to assess activation of relevant signaling pathways

• Animal Model Studies

  • Develop xenograft models with modified C8orf34 expression

  • Use C8orf34 antibodies in IHC analysis of tumor tissues

  • Correlate C8orf34 expression with tumor growth parameters and metastatic potential