C22orf39 Antibody, Biotin conjugated

What is C22orf39 and why is it important in research?

C22orf39 (Chromosome 22 Open Reading Frame 39) is a protein encoded by a gene located on human chromosome 22. This protein is also known as UPF0545 protein C22orf39 at the protein level, with homologs across various species . Interest in C22orf39 has increased due to its presence within the 22q11.2 chromosomal region, which is linked to schizophrenia risk when deleted . Research has shown that the Drosophila ortholog of C22orf39 (CG15908) affects sleep regulation when knocked down in the nervous system, suggesting potential neurological functions . Methodologically, studying C22orf39 through specific antibodies allows researchers to investigate its expression patterns and potential roles in neuropsychiatric disorders.

What experimental applications are appropriate for biotin-conjugated C22orf39 antibodies?

Biotin-conjugated C22orf39 antibodies are particularly suited for several experimental applications:

• Western Blotting (WB): These antibodies can detect C22orf39 protein in complex protein mixtures, allowing for size determination and semi-quantitative analysis .

• ELISA: The biotin conjugation enhances detection sensitivity in enzyme-linked immunosorbent assays, allowing for quantification of C22orf39 in samples .

• Immunohistochemistry (IHC): Particularly useful for frozen (IHC-fro) and paraffin-embedded (IHC-p) tissue sections, enabling visualization of C22orf39 distribution in tissues .

When designing experiments, researchers should consider that C22orf39 antibodies with biotin conjugation targeting amino acids 31-105 have demonstrated reactivity with human samples across these applications .

What controls should be included when using C22orf39 biotin-conjugated antibodies?

For methodological rigor, the following controls should be implemented:

How should researchers optimize Western blot protocols for biotin-conjugated C22orf39 antibodies?

Optimization for Western blotting with biotin-conjugated C22orf39 antibodies requires careful attention to several methodological factors:

• Sample Preparation: Use protein extraction buffers containing protease inhibitors to prevent degradation of C22orf39.

• Protein Loading: Load 20-50 μg of total protein per lane, depending on expression levels.

• Transfer Conditions: For optimal transfer of C22orf39, use PVDF membranes with 0.45 μm pore size.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature.

• Antibody Dilution: Start with a 1:500 to 1:1000 dilution of the C22orf39 biotin-conjugated antibody.

• Detection System: Use streptavidin-HRP conjugate rather than secondary antibodies, leveraging the strong biotin-streptavidin interaction.

• Exposure Time: Begin with short exposures (30 seconds) and increase as needed to visualize bands.

Since C22orf39 antibodies are purified by Protein A , they typically yield clean results with minimal background when these parameters are properly optimized.

What are the advantages and limitations of using biotin-conjugated versus unconjugated C22orf39 antibodies?

Comparing biotin-conjugated and unconjugated C22orf39 antibodies reveals distinct methodological considerations:

Advantages of Biotin Conjugation:

• Enhanced signal amplification through the biotin-streptavidin system

• Compatibility with multiple detection methods (colorimetric, fluorescent, chemiluminescent)

• Reduced background in multi-color immunofluorescence experiments

• Streamlined protocols by eliminating secondary antibody incubation steps

• Greater sensitivity in low-abundance protein detection

Limitations of Biotin Conjugation:

• Potential interference with endogenous biotin in certain tissues

• Limited ability to modify signal strength through secondary antibody dilution adjustments

• Restricted flexibility in detection system selection

• Possible steric hindrance affecting epitope recognition if biotin is conjugated near the antigen-binding site

• Additional cost compared to unconjugated antibodies

When designing complex experiments, researchers should consider that unconjugated C22orf39 antibodies offer broader application flexibility (including IF in cultured cells and paraffin sections) , while biotin-conjugated versions excel in specific applications requiring signal amplification.

How can C22orf39 biotin-conjugated antibodies be effectively used in immunohistochemistry studies?

For effective immunohistochemistry with biotin-conjugated C22orf39 antibodies, follow this methodological framework:

• Tissue Preparation:

  • For frozen sections: Fix in cold acetone for 10 minutes

  • For paraffin sections: Perform heat-mediated antigen retrieval using citrate buffer (pH 6.0)

• Endogenous Biotin Blocking:

  • Critical step: Use an avidin/biotin blocking kit before antibody application

  • Apply avidin solution for 15 minutes, wash, then apply biotin solution for 15 minutes

• Primary Antibody Application:

  • Dilute C22orf39 biotin-conjugated antibody (targeting aa 31-105) at 1:50-1:200

  • Incubate overnight at 4°C in a humidified chamber

• Detection:

  • Apply streptavidin-HRP (1:500) for 30 minutes at room temperature

  • Develop with DAB substrate for 2-5 minutes, monitoring color development

  • Counterstain with hematoxylin for nuclear visualization

• Controls:

  • Include sections from tissues with known C22orf39 expression

  • Prepare negative controls by omitting primary antibody

This approach maximizes sensitivity while minimizing background staining, particularly important when studying C22orf39 in neurological tissues relevant to the 22q11.2 deletion syndrome .

What is the recommended protocol for using C22orf39 biotin-conjugated antibodies in ELISA?

For ELISA applications with biotin-conjugated C22orf39 antibodies, the following detailed protocol is recommended:

• Plate Coating:

  • Coat high-binding 96-well plates with capture antibody against C22orf39 (non-biotin conjugated, targeting a different epitope)

  • Use 100 μL/well of antibody at 1-10 μg/mL in carbonate/bicarbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

• Blocking:

  • Block with 300 μL/well of 1-5% BSA in PBS for 1-2 hours at room temperature

• Sample Application:

  • Add 100 μL/well of diluted samples and standards

  • Incubate for 2 hours at room temperature with gentle shaking

• Detection Antibody:

  • Add 100 μL/well of biotin-conjugated C22orf39 antibody (1:1000 dilution)

  • Incubate for 1 hour at room temperature

• Signal Development:

  • Add 100 μL/well of streptavidin-HRP (1:5000 dilution)

  • Incubate for 30 minutes at room temperature

  • Wash and add 100 μL/well of TMB substrate

  • Stop reaction with 50 μL/well of 2N H₂SO₄ after 15-30 minutes

• Analysis:

  • Read absorbance at 450 nm with 570 nm reference

  • Calculate concentrations using standard curve

This sandwich ELISA approach provides high specificity and sensitivity for C22orf39 detection, with the biotin-conjugated antibody serving as the detection antibody .

How can C22orf39 antibodies contribute to research on schizophrenia and the 22q11.2 deletion syndrome?

C22orf39 antibodies provide critical tools for investigating the neurobiological underpinnings of 22q11.2 deletion syndrome and associated schizophrenia risk. Methodological approaches include:

• Expression Analysis in Patient Samples:

  • Biotin-conjugated C22orf39 antibodies can be used for immunohistochemical analysis of post-mortem brain tissue from patients with 22q11.2 deletion syndrome or schizophrenia

  • Western blotting to quantify expression differences between patient and control samples

• Functional Studies in Model Systems:

  • Following the approach used in Drosophila studies, where knockdown of the C22orf39 ortholog (CG15908) increased total sleep in males

  • Combining antibody-based protein detection with behavioral assessments to correlate protein levels with phenotypes

• Interaction Studies:

  • Immunoprecipitation using C22orf39 antibodies to identify protein-protein interactions that may be disrupted in 22q11.2 deletion syndrome

  • Co-localization studies with other proteins implicated in the syndrome

• Cellular Pathway Analysis:

  • Similar to how researchers examined LZTR1's interaction with Nf1 in Ras signaling and GABA receptor function , C22orf39 can be studied for its potential role in neuronal signaling pathways

These methodological approaches leverage the specificity of C22orf39 antibodies to provide insight into how this protein might contribute to the neuropsychiatric phenotypes associated with the 22q11.2 deletion .

What considerations should be made when using C22orf39 antibodies in cross-species research?

When applying C22orf39 antibodies across different species, researchers must address several methodological challenges:

• Epitope Conservation Analysis:

  • Compare sequence homology of the antibody's target region (amino acids 31-105 in human C22orf39) across species

  • The available C22orf39 antibodies are specifically validated for human reactivity , but orthologous proteins exist in other species

• Validation Strategy for Non-human Samples:

  • Perform Western blot analysis with positive controls from human samples alongside the species of interest

  • Include recombinant proteins from different species (available as yeast-expressed proteins for cow, orangutan, and Xenopus laevis)

• Modified Protocol Parameters:

  • Increase antibody concentration (typically 1.5-2x) when testing in non-validated species

  • Extend primary antibody incubation time to enhance weak cross-reactivity

• Alternative Approaches:

  • For Drosophila studies, consider antibodies against CG15908 rather than direct use of human C22orf39 antibodies

  • Use recombinant expression of tagged C22orf39 orthologs when direct detection is challenging

• Cross-reactivity Documentation:

  • Maintain detailed records of cross-reactivity testing results, including both positive and negative findings

  • Document specific protocol modifications required for each species

While the commercial C22orf39 antibodies are primarily validated for human samples, the availability of recombinant proteins from multiple species suggests the potential for broader applications with appropriate validation .

How can researchers accurately quantify C22orf39 protein levels in complex biological samples?

Accurate quantification of C22orf39 in complex samples requires methodological rigor and multiple complementary approaches:

• Quantitative Western Blotting:

  • Prepare standard curves using recombinant C22orf39 protein at known concentrations

  • Use fluorescently labeled streptavidin rather than chemiluminescence for more linear signal response

  • Apply image analysis software with background subtraction and normalization to housekeeping proteins

• ELISA-Based Quantification:

  • Develop a standard curve using recombinant C22orf39 protein (5-500 ng/mL range)

  • Ensure sample dilutions fall within the linear range of the standard curve

  • Calculate concentrations accounting for dilution factors and normalize to total protein

• Mass Spectrometry Validation:

  • Complement antibody-based detection with targeted mass spectrometry

  • Use immunoprecipitation with C22orf39 antibodies followed by mass spectrometry for enhanced specificity

  • Identify unique peptides from C22orf39 for absolute quantification

• Sample Preparation Considerations:

  • Extract proteins using buffers optimized for nuclear and membrane proteins

  • Include protease inhibitors to prevent degradation

  • Standardize protein extraction efficiency across samples

• Data Analysis Framework:

  • Apply statistical methods appropriate for the dynamic range of the assay

  • Use technical and biological replicates to assess measurement variability

  • Report results with appropriate confidence intervals

This multi-faceted approach enhances reliability when quantifying C22orf39, particularly in neurological samples where expression may be regionally variable and potentially altered in conditions like schizophrenia .

What are common technical challenges when working with C22orf39 biotin-conjugated antibodies and how can they be addressed?

Researchers frequently encounter specific technical challenges when working with biotin-conjugated C22orf39 antibodies. Here are methodological solutions:

Additionally, when working with neurological samples relevant to 22q11.2 deletion syndrome research, tissue handling becomes critical as post-mortem changes can significantly affect epitope recognition .

How should researchers validate the specificity of C22orf39 biotin-conjugated antibodies?

Comprehensive validation of C22orf39 biotin-conjugated antibodies should follow this methodological framework:

• Positive Control Testing:

  • Confirm reactivity using recombinant human C22orf39 protein

  • Test human tissue samples with known C22orf39 expression

• Negative Control Analysis:

  • Examine samples from C22orf39 knockout models or cell lines

  • Use siRNA knockdown of C22orf39 to create control samples with reduced expression

• Cross-reactivity Assessment:

  • Test against closely related proteins in the same family

  • Evaluate potential cross-reactivity with other biotin-containing proteins

• Peptide Blocking Experiments:

  • Pre-incubate antibody with the immunizing peptide (from human C22orf39 aa 31-105)

  • Demonstrate signal elimination when the antibody is neutralized

• Multiple Detection Methods:

  • Confirm findings using alternative techniques beyond Western blotting

  • Compare results from biotin-conjugated antibody with unconjugated version

• Replicate with Alternative Antibodies:

  • Validate key findings using antibodies targeting different C22orf39 epitopes

  • Compare results with antibodies from different manufacturers when available

This systematic validation approach ensures reliable results, particularly important when investigating C22orf39 in the context of neuropsychiatric research where tissue-specific expression patterns may be subtle and biologically significant .

How might C22orf39 antibodies be used to investigate potential roles in sleep regulation based on recent Drosophila studies?

The discovery that C22orf39's Drosophila ortholog (CG15908) affects sleep regulation opens several methodological avenues for investigation using biotin-conjugated C22orf39 antibodies:

• Comparative Expression Analysis:

  • Map C22orf39 expression in sleep-regulating regions of mammalian brains

  • Compare expression patterns between nocturnal and diurnal animals

  • Examine expression changes during sleep-wake cycles using time-course sampling

• Co-localization Studies:

  • Perform double-labeling with markers of specific neuronal populations

  • Investigate potential co-expression with GABA-responsive neurons and Rdl receptors, given the Drosophila findings

  • Examine relationship with Nf1 and Cul3, which showed genetic interactions with CG15908 in Drosophila

• Circuit-Level Investigation:

  • Use biotin-conjugated antibodies in conjunction with neural circuit tracing

  • Examine C22orf39 expression in circuits known to regulate sleep homeostasis

• Functional Correlation Analysis:

  • Correlate C22orf39 protein levels with sleep parameters in model organisms

  • Examine expression changes in response to sleep deprivation

• Translational Research Approach:

  • Investigate C22orf39 expression in patients with sleep disturbances, particularly those with 22q11.2 deletion syndrome

  • Correlate protein levels with polysomnography data

This methodological framework leverages the specificity of biotin-conjugated C22orf39 antibodies to build upon the Drosophila findings that suggested C22orf39 may play a conserved role in sleep regulation across species .

What considerations should be made when designing multi-omics studies incorporating C22orf39 antibody-based detection?

Integrating C22orf39 antibody-based detection into multi-omics research requires careful methodological planning:

• Sample Processing Harmonization:

  • Develop protocols allowing simultaneous extraction of proteins, RNA, and DNA

  • Standardize tissue handling to maintain compatibility across platforms

  • Consider how fixation affects both antibody epitopes and nucleic acid integrity

• Correlative Analysis Framework:

  • Design experiments to correlate C22orf39 protein levels (using biotin-conjugated antibodies) with:

    • Transcriptomic data (C22orf39 mRNA expression)

    • Genomic data (copy number variation, mutations in 22q11.2 region)

    • Epigenomic data (methylation status of C22orf39 promoter)

• Temporal Considerations:

  • Account for potential time lags between transcriptional and translational changes

  • Design time-course experiments capturing dynamic regulation

• Spatial Resolution Integration:

  • Combine single-cell RNA sequencing with immunohistochemistry using biotin-conjugated C22orf39 antibodies

  • Develop protocols for spatial transcriptomics with antibody-based protein mapping

• Data Integration Strategy:

  • Implement computational methods specifically designed for protein-RNA correlation

  • Develop normalization strategies across different data types

  • Apply machine learning approaches to identify patterns across multi-omics datasets

• Validation Design:

  • Plan for orthogonal validation of key findings

  • Include technical replicates across platforms to assess technology-specific variability

This methodological framework enables researchers to place C22orf39 protein expression in broader biological context, particularly valuable when investigating its potential role in complex phenotypes associated with the 22q11.2 deletion syndrome .

What are the key methodological considerations for researchers planning studies with C22orf39 biotin-conjugated antibodies?

Researchers planning studies with C22orf39 biotin-conjugated antibodies should prioritize these methodological considerations:

• Application-Specific Optimization:

  • Select appropriate antibody concentration depending on the application (typically 1:50-1:200 for immunohistochemistry, 1:500-1:1000 for Western blotting)

  • Validate the specific biotin-conjugated antibody for your application before conducting full experiments

• Experimental Controls:

  • Include comprehensive controls to validate specificity and reliability

  • Consider both technical controls (no primary antibody) and biological controls (tissues with known expression patterns)

• Detection System Selection:

  • Choose streptavidin conjugates appropriate for the desired detection method

  • Consider signal amplification needs based on expected abundance of C22orf39

• Cross-Species Applications:

  • Validate antibodies when working with non-human samples, as most C22orf39 antibodies are primarily validated for human reactivity

  • Consider using recombinant proteins from the species of interest as positive controls

• Context-Specific Interpretation:

  • Interpret findings in light of the current understanding of C22orf39 function

  • Consider the potential role in neurological processes suggested by Drosophila studies