C11orf1 Antibody

What is C11orf1 and why is it significant in research?

C11orf1, also known as chromosome 11 open reading frame 1 or UPF0686 protein C11orf1, is a protein believed to play a role in cell cycle progression and potentially in tumorigenesis. It belongs to the UPF0686 family and is primarily localized in the nucleoplasm and nucleus . With a molecular weight of approximately 17,785 Da, this protein is of interest in cancer research due to its potential implications in cell growth regulation and cancer development . Understanding C11orf1's function is crucial for investigating molecular mechanisms underlying oncogenesis and potentially developing targeted cancer therapies.

What experimental applications have C11orf1 antibodies been validated for?

C11orf1 antibodies have been validated for multiple experimental applications, primarily:

• Western blot (WB): For detecting denatured C11orf1 protein in cell and tissue lysates

• Enzyme-linked immunosorbent assay (ELISA): For quantitative detection of C11orf1, typically at dilutions of 1:2000-1:10000

• Immunohistochemistry (IHC): For examining C11orf1 expression in paraffin-embedded tissues, recommended at dilutions of 1:20-1:200

• Immunocytochemistry-immunofluorescence (ICC-IF): For visualizing cellular localization patterns

When designing experiments, researchers should consider that most commercially available C11orf1 antibodies have been primarily validated against human samples .

What protocols should be followed for immunohistochemical detection of C11orf1?

For optimal immunohistochemical detection of C11orf1 in paraffin-embedded tissues:

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

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Apply C11orf1 primary antibody at the recommended dilution (typically 1:20-1:200)

• Incubate overnight at 4°C in a humidified chamber

• Apply appropriate secondary antibody (e.g., HRP-conjugated anti-rabbit IgG)

• Develop with DAB substrate and counterstain with hematoxylin

• Include positive control tissues with known C11orf1 expression (brain and kidney tissues have been validated)

This protocol has been successfully used to detect C11orf1 in human brain and kidney tissues at a dilution of 1:100 .

What are the recommended storage conditions for C11orf1 antibodies?

To maintain antibody functionality:

• Store C11orf1 antibodies at -20°C for long-term storage

• For frequent use, aliquot to avoid repeated freeze-thaw cycles (more than 3 cycles can diminish activity)

• When refrigerated, store at 4°C for up to two weeks

• Protect from light exposure, particularly for conjugated antibodies

• Ensure the glycerol content (typically 50%) prevents freezing damage

• Monitor pH stability (typically in a storage buffer of 0.01M PBS, pH 7.4)

Proper storage is crucial as antibody degradation can lead to non-specific binding and unreliable results in subsequent experiments.

How can researchers validate the specificity of C11orf1 antibodies?

Rigorous validation of C11orf1 antibodies is essential given the known issues with antibody specificity in the research community . A comprehensive validation approach should include:

• Positive and negative controls: Use tissues/cells with known high and low/absent C11orf1 expression

• Knockout/knockdown validation: Compare staining between wild-type samples and those with C11orf1 knockdown or knockout

• Peptide competition assay: Pre-incubate antibody with immunogen peptide to block specific binding

• Multiple antibody comparison: Test different antibody clones targeting distinct epitopes of C11orf1

• Western blot correlation: Confirm that band size matches expected molecular weight (17,785 Da)

• Orthogonal validation: Compare protein expression with mRNA levels using qPCR

• Cross-reactivity testing: Test on samples from species not covered by the antibody's reactivity profile

This multi-faceted approach addresses the significant problem that "fifty percent of the antibodies on the market are junk" and may bind to unintended targets .

What methodological considerations are important when using C11orf1 antibodies in Western blotting?

When optimizing Western blot protocols for C11orf1 detection:

• Sample preparation: Use RIPA or NP-40 buffer with protease inhibitors; include both nuclear and cytoplasmic fractions due to C11orf1's nuclear localization

• Protein loading: Load 20-40μg of total protein per lane

• Gel selection: Use 12-15% SDS-PAGE gels due to C11orf1's relatively small size (17.8 kDa)

• Transfer conditions: Optimize for small proteins (higher voltage for shorter time)

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute according to manufacturer recommendations in blocking buffer

• Incubation time: Overnight at 4°C with gentle rocking

• Chemiluminescence detection: Use high-sensitivity ECL substrate for optimal signal-to-noise ratio

• Controls: Include recombinant C11orf1 protein as positive control

These considerations help address typical challenges in detecting small nuclear proteins like C11orf1.

How can researchers investigate potential protein-protein interactions involving C11orf1?

To elucidate C11orf1's functional network:

• Co-immunoprecipitation (Co-IP): Use C11orf1 antibodies to pull down protein complexes, followed by mass spectrometry to identify interacting partners

• Proximity ligation assay (PLA): Visualize and quantify protein interactions in situ using C11orf1 antibody paired with antibodies against suspected interaction partners

• Yeast two-hybrid screening: Use C11orf1 as bait to screen for potential interactors

• Bimolecular fluorescence complementation (BiFC): Visualize direct interactions in living cells

• FRET/FLIM analysis: Measure energy transfer between fluorescently labeled C11orf1 and potential partners

• Cross-linking mass spectrometry: Identify direct binding interfaces

• Immunofluorescence co-localization: Examine spatial relationships with other proteins, particularly in the nucleoplasm

These approaches can help determine C11orf1's role in protein complexes that may regulate cell cycle progression.

What strategies can address batch-to-batch variability in C11orf1 antibody performance?

Batch-to-batch variability represents a significant challenge in antibody-based research, potentially leading to irreproducible results . To mitigate this issue:

• Purchase larger lots: When possible, secure sufficient antibody from a single lot for entire research projects

• Perform lot-specific validation: Validate each new lot against your specific application before use

• Create internal standards: Generate reference samples with known C11orf1 expression for comparison across experiments

• Consider recombinant antibodies: These offer better consistency than traditional polyclonal antibodies

• Implement standardized protocols: Document detailed procedures to minimize technical variability

• Use multiple detection methods: Confirm key findings with orthogonal approaches

• Consider alternative technologies: Explore CRISPR-based protein tagging approaches for consistent protein labeling

As noted in the fourth search result, "It's thought that these bad antibodies lead to a large fraction of the irreproducible results... Say you have a perfect antibody that binds exactly what you want and nothing else. And then you order the next lot and there's a different preparation from a different animal, and you're back to square one."

How can researchers optimize C11orf1 detection in challenging tissue types?

When working with tissues that present detection challenges:

• Optimize fixation protocols: Test different fixation times and conditions (4-24 hours in 10% neutral buffered formalin)

• Enhance antigen retrieval: Compare heat-mediated retrieval methods (citrate, EDTA, Tris-EDTA buffers) at various pH levels

• Employ signal amplification: Use tyramide signal amplification or polymer-based detection systems

• Test epitope exposure techniques: Include detergent permeabilization steps or protease digestion

• Adjust blocking conditions: Test various blocking agents (BSA, normal serum, commercial blockers) to reduce background

• Optimize antibody concentration: Perform titration experiments with dilutions from 1:20 to 1:200

• Extend incubation times: Consider longer primary antibody incubations (24-48 hours at 4°C)

• Use chromogenic multiplex IHC: To differentiate C11orf1 from other nuclear proteins

These approaches have been successful in detecting C11orf1 in various human tissues, including brain and kidney samples .

What experimental designs are appropriate for investigating C11orf1's potential role in cancer?

To explore C11orf1's involvement in tumorigenesis:

• Expression profiling: Compare C11orf1 levels between tumor and matched normal tissues using IHC and Western blotting

• Correlation analysis: Analyze associations between C11orf1 expression and clinical parameters (stage, grade, survival)

• Functional studies: Use RNA interference or CRISPR to modulate C11orf1 expression in cancer cell lines, followed by:

  • Proliferation assays

  • Cell cycle analysis

  • Apoptosis assays

  • Migration/invasion assays

• Mechanistic investigation: Determine if C11orf1 interacts with known oncogenes or tumor suppressors

• Animal models: Examine effects of C11orf1 overexpression or knockout in tumor xenograft models

• Multi-omics integration: Correlate C11orf1 protein levels with genomic and transcriptomic data

Since C11orf1 is "believed to play a role in cell cycle progression and may be involved in tumorigenesis," these approaches can help clarify its potential as a biomarker or therapeutic target .

How do researchers analyze discrepancies in C11orf1 expression data across different experimental platforms?

When faced with conflicting results:

• Platform-specific considerations:

  • Western blot: Evaluate antibody specificity, sample preparation, and loading controls

  • IHC: Assess fixation effects, antigen retrieval methods, and scoring systems

  • RNA quantification: Compare with protein levels to identify post-transcriptional regulation

• Systematic analysis approach:

  • Create a standardized experimental pipeline to minimize technical variables

  • Use multiple antibodies targeting different epitopes of C11orf1

  • Compare polyclonal (broader epitope recognition) vs. monoclonal (higher specificity) results

  • Implement positive and negative controls for each platform

  • Consider cell/tissue heterogeneity in bulk sample analyses

• Data integration methods:

  • Weighted averaging based on technique reliability

  • Bayesian integration of multi-platform data

  • Meta-analysis approaches for cross-study comparison

This methodical approach helps resolve conflicting data and addresses the known issue that "fifty percent of the antibodies on the market are junk. They're not specific."

What are the current limitations in C11orf1 research and how might they be addressed?

Current limitations and potential solutions include:

• Incomplete protein characterization:

  • Solution: Apply structural biology approaches (X-ray crystallography, cryo-EM) to determine C11orf1 structure

  • Develop better computational models of protein function

• Limited known interactors:

  • Solution: Perform comprehensive interactome studies using proximity labeling (BioID, APEX)

  • Apply systematic protein complex purification followed by mass spectrometry

• Specificity challenges with current antibodies:

  • Solution: Develop more specific monoclonal antibodies targeting unique epitopes

  • Explore alternative protein labeling technologies, such as CRISPR-based tagging as mentioned in the fourth search result

• Unclear physiological function:

  • Solution: Generate knockout models (cells, organoids, animals) for phenotypic characterization

  • Perform rescue experiments with wild-type and mutant forms of C11orf1

• Limited disease association data:

  • Solution: Conduct larger-scale analysis across tumor databases (TCGA, ICGC)

  • Investigate potential roles in non-cancer pathologies

Addressing these limitations will advance understanding of C11orf1's biological significance and potential therapeutic applications.