C5orf24 Antibody

What is C5orf24 and why is it studied?

C5orf24 (chromosome 5 open reading frame 24) is a protein encoded by the C5orf24 gene with ID 134553. It functions as a cellular transcriptional regulator that performs different functions in different cell types . The protein has a calculated molecular weight of approximately 20 kDa, though it is typically observed at 23 kDa in experimental settings . C5orf24 is studied to understand its role in cellular regulation and potential involvement in various biological processes. The UniProt ID for this protein is Q7Z6I8, with several secondary accession numbers including D3DQA7, Q86Y53, and Q8N1T9 .

What types of C5orf24 antibodies are available for research?

• HRP-conjugated (for enhanced ELISA applications)

• FITC-conjugated (for fluorescence applications)

• Biotin-conjugated (for various detection systems)

Specialized options such as the Prestige Antibodies from Sigma-Aldrich are designed for specific applications like immunofluorescence and immunohistochemistry .

What applications can C5orf24 antibodies be used for?

C5orf24 antibodies can be used for multiple experimental applications including:

The antibodies have been validated particularly well for Western blot applications, with published literature supporting their use in this technique .

What are the optimal conditions for Western blotting with C5orf24 antibodies?

For optimal Western blotting with C5orf24 antibodies, follow these methodological guidelines:

• Sample preparation: Use HEK-293 cells as a positive control, as these have been extensively validated .

• Loading amount: 20-30 μg of total protein is typically sufficient.

• Dilution range: Start with 1:1000 dilution and adjust as needed within the 1:500-1:5000 range .

• Expected band size: Look for a band at approximately 23 kDa (observed molecular weight) .

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

• Incubation time: Primary antibody incubation overnight at 4°C, followed by appropriate secondary antibody (anti-rabbit IgG) incubation for 1 hour at room temperature.

• Detection system: Both chemiluminescence and fluorescence detection systems are compatible.

Remember that optimizing antibody dilution for each specific experimental setup and sample type is crucial for achieving the best signal-to-noise ratio .

How should I prepare samples for immunohistochemistry with C5orf24 antibodies?

For immunohistochemistry applications with C5orf24 antibodies, sample preparation is critical:

• Fixation: Use 4% paraformaldehyde or 10% neutral buffered formalin.

• Antigen retrieval: Perform heat-induced epitope retrieval using TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 as an alternative .

• Blocking: Use 5-10% normal serum (matching the species of the secondary antibody) with 1% BSA.

• Antibody dilution: Start with 1:100 dilution and adjust within the 1:50-1:500 range based on signal intensity .

• Human tissues validated: Pancreas and kidney tissues have been confirmed to show positive staining .

• Incubation conditions: Incubate primary antibody overnight at 4°C or 1-2 hours at room temperature, followed by appropriate detection system.

Each new tissue type may require optimization of antigen retrieval methods and antibody concentrations .

What controls should I include when using C5orf24 antibodies?

When designing experiments with C5orf24 antibodies, include these essential controls:

• Positive control: HEK-293 cells have been extensively validated for Western blot, immunofluorescence, immunoprecipitation, and flow cytometry applications .

• Tissue controls: Human pancreas and kidney tissues are recommended for immunohistochemistry .

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Blocking peptide/antigen pre-absorption control when available

• Loading control: Include appropriate housekeeping proteins for Western blots (β-actin, GAPDH, or tubulin).

• Molecular weight marker: Always include to confirm the expected 23 kDa size .

These controls help validate antibody specificity and ensure experimental rigor by differentiating between true signal and background or non-specific binding.

How can I confirm C5orf24 antibody specificity and rule out cross-reactivity?

Confirming antibody specificity is crucial for reliable research results. For C5orf24 antibodies:

• Knockdown/knockout validation: Generate C5orf24 knockdown (siRNA, shRNA) or knockout (CRISPR/Cas9) cells and confirm loss of signal. Specific siRNA and shRNA products are available targeting C5orf24 .

• Overexpression validation: Compare signal in cells overexpressing C5orf24 versus control cells.

• Peptide competition assay: Pre-incubate the antibody with purified C5orf24 protein or immunogen peptide (Ag19534 for Proteintech antibody) .

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of C5orf24 and compare staining patterns.

• Orthogonal techniques: Correlate antibody-based detection with mRNA expression levels via RT-PCR.

• Species cross-reactivity testing: While primarily reactive with human C5orf24, cross-reactivity has been cited with pig samples . For broader cross-species studies, certain antibodies claim reactivity with bat, chicken, cow, dog, guinea pig, hamster, horse, monkey, mouse, rabbit, rat, and Xenopus laevis samples .

Through these rigorous validation approaches, you can confidently establish the specificity of C5orf24 antibody signal in your experimental system.

What is the best approach for troubleshooting weak or absent signal in C5orf24 immunodetection?

When facing challenges with weak or absent C5orf24 detection, follow this systematic troubleshooting approach:

• Expression level assessment:

  • Verify if your cell line/tissue expresses C5orf24 (HEK-293 cells are confirmed to express detectable levels) .

  • Consider concentration/enrichment steps like immunoprecipitation before detection.

• Sample preparation optimization:

  • For Western blot: Ensure complete protein denaturation; add protease inhibitors to prevent degradation.

  • For IHC/IF: Test multiple antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0) .

• Antibody considerations:

  • Decrease dilution (use more concentrated antibody) within recommended ranges .

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

  • Try fresh antibody aliquot (avoid repeated freeze-thaw cycles).

  • Consider different antibody clone or vendor if persistent issues occur.

• Detection system enhancement:

  • Use more sensitive detection methods (ECL-Plus, tyramide signal amplification).

  • For fluorescence applications, consider longer exposure times or more sensitive cameras.

  • For IHC, try polymer-based detection systems for signal amplification.

• Blocking and washing optimization:

  • Decrease blocking stringency (reduce blocking reagent concentration).

  • Reduce washing stringency (decrease salt concentration or washing time).

For Western blot specifically, remember that C5orf24 has an observed molecular weight of 23 kDa, which may run differently depending on gel percentage and buffer systems .

How can I optimize multiplexed detection involving C5orf24 antibodies?

For multiplexed detection experiments involving C5orf24 with other targets:

• Primary antibody compatibility:

  • Select C5orf24 antibodies raised in rabbit and pair with antibodies raised in different species (mouse, goat) for other targets.

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

• Fluorescence multiplexing (IF/ICC):

  • Utilize C5orf24 antibodies at 1:200-1:800 dilution .

  • Choose fluorophores with minimal spectral overlap.

  • Perform appropriate controls including single-color staining to assess bleed-through.

  • Consider spectral unmixing for challenging combinations.

• Chromogenic multiplexing (IHC):

  • Use enzyme systems with distinct chromogens (HRP/DAB for C5orf24, AP/Fast Red for second target).

  • Optimize antibody dilutions (1:50-1:500 for C5orf24) .

  • Consider automated multiplexing platforms for consistent results.

• Flow cytometry multiplexing:

  • Use C5orf24 at 0.40 μg per 10^6 cells .

  • Proper compensation and FMO (fluorescence minus one) controls are essential.

  • Consider intracellular staining protocols as C5orf24 is an intracellular target.

• Western blot multiplexing:

  • For fluorescence-based detection, use differentially labeled secondary antibodies.

  • For chemiluminescence, sequential probing with thorough stripping between targets is recommended.

  • If targets have similar molecular weights, consider using differentially labeled primary antibodies.

These approaches enable simultaneous detection of C5orf24 alongside other proteins of interest while maintaining specificity and minimizing cross-reactivity.

How should I interpret variations in C5orf24 molecular weight across different experimental systems?

Researchers often observe variations in C5orf24 molecular weight, which can be interpreted as follows:

• Expected weight parameters:

  • Calculated molecular weight: 20 kDa (188 amino acids)

  • Typically observed molecular weight: 23 kDa

• Common causes for molecular weight variations:

  • Post-translational modifications: Look for evidence of phosphorylation, glycosylation, or other modifications.

  • Alternative splicing: Multiple transcript variants may exist producing proteins of different lengths.

  • Experimental conditions: Different sample preparation methods, buffer systems, or gel percentages can affect protein migration.

  • Cell/tissue-specific modifications: Protein modifications may vary between different biological contexts.

• Analytical approach for weight variations:

  • Compare to positive control (HEK-293 cells) run simultaneously .

  • Use mass spectrometry to confirm protein identity if significant deviations occur.

  • Consider 2D gel electrophoresis to separate potential isoforms or modified versions.

  • Treat samples with phosphatases or glycosidases to identify contribution of these modifications.

When reporting results, always note both the expected theoretical weight (20 kDa) and your observed weight, explaining any discrepancies based on the considerations above .

What are the best practices for quantifying C5orf24 expression levels across different experimental techniques?

For accurate quantification of C5orf24 expression:

• Western Blot quantification:

  • Use appropriate loading controls (housekeeping proteins) for normalization.

  • Ensure signal is within linear range of detection.

  • Apply densitometry analysis using software like ImageJ.

  • Present data as normalized relative expression (C5orf24/loading control ratio).

  • Recommended dilution: 1:500-1:2000 .

• Immunohistochemistry quantification:

  • Use digital image analysis for objective scoring.

  • Quantify percentage of positive cells and staining intensity.

  • Develop consistent scoring system (H-score or Allred score).

  • Include reference standards in each experiment.

  • Recommended dilution: 1:50-1:500 .

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity within defined cellular regions.

  • Use consistent acquisition parameters between samples.

  • Present data as normalized to cell area or nuclear counterstain.

  • Recommended dilution: 1:200-1:800 .

• Flow cytometry quantification:

  • Report mean/median fluorescence intensity.

  • Use appropriate statistical transformations for flow data.

  • Include quantification beads for absolute quantification when needed.

  • Recommended concentration: 0.40 μg per 10^6 cells .

• Cross-technique validation:

  • Validate findings across multiple techniques.

  • Correlate protein detection with mRNA expression when possible.

  • Consider absolute quantification methods for more precise measurements.

When presenting quantitative data, always include statistical analysis with appropriate tests depending on data distribution and experimental design.

How can I reconcile contradictory findings between different detection methods when studying C5orf24?

When faced with contradictory results between different detection methods:

• Application-specific considerations:

  • WB detects denatured proteins, while IF/IHC detect proteins in their native conformation, potentially explaining differences in antibody recognition.

  • Flow cytometry detects intracellular C5orf24 and requires effective permeabilization .

  • IP studies isolate C5orf24 along with interaction partners, potentially affecting apparent molecular weight or detection.

• Methodological reconciliation approach:

  • Control standardization: Ensure positive controls (HEK-293 cells) and negative controls are consistent across methods .

  • Epitope accessibility: Consider whether the epitope recognized by the antibody might be masked in certain applications or biological contexts.

  • Antibody validation: Confirm antibody specificity in each application separately, as performance can vary by method .

  • Subcellular localization: Assess whether discrepancies reflect differences in detecting C5orf24 in different cellular compartments.

• Technical resolution strategies:

  • Use multiple antibodies targeting different epitopes of C5orf24.

  • Implement orthogonal detection methods (mass spectrometry, RNA-seq).

  • Employ genetic approaches (overexpression, knockdown) to validate antibody specificity in each system.

  • Develop a comprehensive model incorporating all findings, accounting for method-specific limitations.

• Documentation and reporting:

  • Thoroughly document all experimental conditions.

  • Report contradictory findings transparently in publications.

  • Provide potential explanations for discrepancies based on methodological differences.

By systematically analyzing contradictory results through these approaches, researchers can develop more comprehensive understanding of C5orf24 biology while accounting for technical limitations of various detection methods.