C10orf53 Antibody

What is C10orf53 and why is it studied?

C10orf53 (chromosome 10 open reading frame 53) is a protein-coding gene that produces a relatively small protein of approximately 93 amino acid residues with a molecular weight of 10.4 kDa. It belongs to the UPF0728 protein family and is notably expressed in testicular tissue. Research interest stems from its potential role in specific tissue functions and as a biomarker in various biological processes . The protein's specific cellular function remains under investigation, making it relevant for researchers studying chromosome 10-associated proteins and their biological significance.

What types of C10orf53 antibodies are available for research?

C10orf53 antibodies are predominantly available as rabbit polyclonal antibodies from several suppliers including Invitrogen, Novus Biologicals, and Abnova . These antibodies are specifically designed to recognize human C10orf53 protein and are provided in various formats including unconjugated and conjugated (such as HRP-conjugated and FITC-conjugated) versions . The diversity in antibody formats supports multiple experimental approaches depending on research requirements and detection methods.

What is the amino acid sequence of the C10orf53 protein targeted by these antibodies?

The human C10orf53 protein has the following amino acid sequence: MPKNAVVILRYGPYSAAGLPVEHHTFRLQGLQAVLAIDGHEVILEKIEDWNVVELMVNEEVIFHCNIKDLEF . Many of the commercially available antibodies are raised against recombinant proteins corresponding to specific regions of this sequence. Understanding this sequence is crucial for researchers aiming to validate antibody specificity and for designing blocking peptides for control experiments.

In which species has C10orf53 been identified?

While the primary research focus remains on human C10orf53, orthologs have been identified in several species including mouse, rat, bovine, frog, zebrafish, and chimpanzee . This cross-species conservation suggests potential evolutionary significance of the protein. Researchers should note that although orthologs exist, most commercially available antibodies are specifically validated for human C10orf53, requiring careful validation for cross-reactivity studies in other organisms.

What are the validated applications for C10orf53 antibodies in research?

C10orf53 antibodies have been validated for multiple applications including:

This range of applications allows researchers to investigate C10orf53 expression and localization in various experimental contexts .

How should researchers determine the optimal antibody dilution for specific applications?

When using C10orf53 antibodies, researchers should conduct preliminary titration experiments using positive and negative controls. For immunohistochemistry applications, start with the recommended dilution range (e.g., 1:50-1:200) and perform a dilution series to determine optimal signal-to-noise ratio. For Western blot applications, optimization should include varying both primary antibody concentration (starting with 0.04-0.4 μg/mL) and incubation conditions (time and temperature). Validation should include appropriate controls such as known positive tissue samples, knockdown/knockout samples, and secondary-only controls to establish specificity.

What detection systems work best with C10orf53 antibodies for immunohistochemistry?

For optimal results in immunohistochemistry, researchers should consider the following detection system approaches:

• For paraffin-embedded sections, heat-induced epitope retrieval methods are recommended, typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) .

• When using polyclonal C10orf53 antibodies, biotin-streptavidin amplification systems provide enhanced sensitivity for detecting low-abundance expression.

• For fluorescent detection, secondary antibodies conjugated to bright fluorophores (Alexa Fluor 488, 594, or 647) are recommended when using unconjugated primary C10orf53 antibodies.

• For direct detection, pre-conjugated antibodies (such as FITC-conjugated variants) eliminate potential cross-reactivity issues in multi-label experiments.

The choice of detection system should be determined by the experimental goal, expression level of C10orf53, and the specific tissue being examined.

What storage conditions maintain optimal C10orf53 antibody activity?

To preserve antibody functionality, store C10orf53 antibodies at 4°C for short-term storage (up to one week) . For long-term storage, -20°C is recommended with the antibody divided into small working aliquots to avoid repeated freeze-thaw cycles . Most commercial preparations are supplied in stabilizing buffers containing glycerol (typically 40-50%) and preservatives such as sodium azide (0.02-0.03%) . Researchers should follow supplier-specific recommendations, as formulations may vary between manufacturers.

How can researchers validate the specificity of C10orf53 antibodies?

Comprehensive validation of C10orf53 antibody specificity should include multiple approaches:

• Western blot analysis demonstrating a single band at the expected molecular weight (approximately 10.4 kDa) , with appropriate positive and negative controls.

• Peptide competition assays using the immunizing peptide or recombinant C10orf53 protein to block specific binding.

• siRNA or CRISPR-mediated knockdown/knockout experiments to confirm reduced or absent signal in target-depleted samples.

• Comparison of staining patterns across multiple antibodies targeting different epitopes of C10orf53.

• Mass spectrometry validation of immunoprecipitated proteins to confirm identity.

Thorough validation is particularly important for C10orf53 given its relatively small size and potential for cross-reactivity with related proteins.

What are the recommended sample preparation methods for detecting C10orf53?

For optimal C10orf53 detection, sample preparation should be tailored to the application:

For Western blot:

• Use RIPA or NP-40 based lysis buffers supplemented with protease inhibitors

• Include reducing agents in sample buffer due to the presence of cysteine residues in the sequence

• Heat samples at 95°C for 5 minutes prior to loading

For Immunohistochemistry:

• For paraffin sections, fixation in 10% neutral-buffered formalin for 24-48 hours is recommended

• Optimal section thickness is 4-6 μm

• Perform antigen retrieval using citrate buffer (pH 6.0) with heating to 95-100°C for 15-20 minutes

For flow cytometry or ICC/IF applications:

• Gentle fixation (2-4% paraformaldehyde for 10-15 minutes)

• Permeabilization with 0.1-0.3% Triton X-100 or 0.1% saponin

These preparation methods should be optimized based on the specific sample type and research question.

How can researchers address non-specific binding when using C10orf53 antibodies?

When facing non-specific binding issues with C10orf53 antibodies, implement these strategic approaches:

• Optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat dry milk, or commercial blocking reagents) and extending blocking time to 1-2 hours.

• Include protein-based carriers (0.1-0.5% BSA) and detergents (0.05-0.1% Tween-20) in antibody dilution buffers.

• For Western blots, perform stringent washing steps (at least 3 × 10 minutes) with TBS-T (0.1% Tween-20).

• Consider pre-adsorption of the antibody with cell/tissue lysates from species that may cause cross-reactivity.

• Titrate both primary and secondary antibodies to determine optimal concentrations that maintain specific signal while minimizing background.

For polyclonal antibodies like those against C10orf53, affinity purification against the immunogen can significantly improve specificity.

What controls are essential when studying C10orf53 expression in different cell types?

Rigorous experimental design for C10orf53 expression studies requires comprehensive controls:

• Positive control tissue/cells: Testis tissue or cell lines with documented C10orf53 expression .

• Negative controls: Tissues/cells known to lack C10orf53 expression or C10orf53 knockout samples.

• Technical controls: Secondary antibody-only controls to assess non-specific binding of detection reagents.

• Isotype controls: Irrelevant antibodies of the same isotype (IgG for polyclonal rabbit antibodies) at equivalent concentrations.

• Biological validation: Correlation of protein detection with mRNA expression data from qPCR or RNA-seq.

• Peptide competition controls: Pre-incubation of the antibody with immunizing peptide to confirm binding specificity.

Implementation of these controls ensures reliable interpretation of C10orf53 expression patterns across different experimental conditions.

How can researchers optimize duplex/multiplex staining protocols involving C10orf53 antibodies?

For successful multiplex staining involving C10orf53 antibodies:

• Select compatible primary antibodies from different host species (avoid rabbit-rabbit combinations with polyclonal C10orf53 antibodies).

• If using multiple rabbit antibodies, consider sequential immunostaining with complete stripping between rounds or use directly conjugated antibodies.

• Implement tyramide signal amplification (TSA) for detecting low-abundance C10orf53 expression while allowing antibody stripping.

• Carefully choose fluorophores with minimal spectral overlap when designing multiplex fluorescence experiments.

• Include appropriate single-stain controls to establish compensation settings for fluorescence detection.

• Validate each antibody individually before combining in multiplex protocols to ensure performance isn't compromised.

These approaches enable simultaneous evaluation of C10orf53 expression alongside other markers of interest in complex tissue architectures.

What approaches can address challenges in detecting low-abundance C10orf53 expression?

When investigating low-level C10orf53 expression, researchers should consider:

• Signal amplification strategies:

  • Biotin-streptavidin amplification systems

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems with multiple HRP molecules

• Sample enrichment approaches:

  • Immunoprecipitation prior to Western blot analysis

  • Tissue microdissection to isolate regions of interest

  • Subcellular fractionation to concentrate compartments with C10orf53 expression

• Detection optimization:

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

  • Increased antibody concentration (while monitoring background)

  • Enhanced chemiluminescence substrates for Western blot

  • Confocal microscopy with increased gain and averaging for IF/IHC

These methodological enhancements can improve detection sensitivity while maintaining specificity for C10orf53.

How can C10orf53 antibodies be integrated into high-throughput screening approaches?

Researchers can leverage C10orf53 antibodies in high-throughput contexts through:

• Tissue microarray (TMA) analysis to evaluate C10orf53 expression across multiple tissues or patient samples simultaneously.

• Automated immunohistochemistry platforms with optimized C10orf53 antibody protocols for consistent staining across large sample cohorts.

• High-content imaging systems for quantitative analysis of C10orf53 subcellular localization and expression levels in cell-based assays.

• Reverse-phase protein arrays (RPPA) for quantitative assessment of C10orf53 across numerous samples with minimal antibody consumption.

• Multiplexed antibody panels incorporating C10orf53 antibodies with other markers for comprehensive phenotyping.

These approaches enable systematic investigation of C10orf53 biology across diverse experimental conditions and sample types.

What is known about potential post-translational modifications of C10orf53 and how might they affect antibody recognition?

While specific post-translational modifications (PTMs) of C10orf53 remain largely uncharacterized, researchers should consider:

• Sequence analysis reveals potential phosphorylation sites within the C10orf53 protein that could affect antibody binding.

• When investigating potential PTMs, researchers should employ phospho-specific or modification-specific antibodies if available.

• For comprehensive PTM analysis, consider combining immunoprecipitation using C10orf53 antibodies with mass spectrometry.

• Different antibodies targeting distinct epitopes may exhibit differential recognition of modified forms of C10orf53.

• Treatment with phosphatases or other enzymatic modifications prior to immunodetection can help determine if antibody recognition is modification-dependent.

Understanding these potential modifications is critical for accurate interpretation of C10orf53 detection patterns in different physiological contexts.

How can researchers contribute to improving C10orf53 antibody validation standards in the field?

To advance C10orf53 antibody validation standards, researchers should:

• Implement comprehensive validation using multiple approaches (Western blot, IHC, IF, flow cytometry) with appropriate controls.

• Document detailed validation protocols, including all experimental conditions, in publications and repositories.

• Utilize genetic knockout models or CRISPR-edited cell lines as definitive controls for antibody specificity.

• Participate in antibody validation initiatives and contribute data to community resources.

• Report both positive and negative findings regarding antibody performance to suppliers and in publications.

• Correlate protein detection with orthogonal measurements (RNA-seq, mass spectrometry) to validate expression patterns.

These collaborative efforts will enhance research reproducibility and accelerate understanding of C10orf53 biology across diverse experimental contexts.