C4orf33 Antibody

What is C4orf33 and what is known about its function?

C4orf33 (Chromosome 4 Open Reading Frame 33) is a poorly characterized protein of 199 amino acids that has been implicated in various cellular processes, including cell proliferation and differentiation. It is classified as UPF0462 protein C4orf33, indicating it belongs to the Uncharacterized Protein Family 0462 .

The protein is encoded by a gene located on human chromosome 4 at position 4q28.2, which spans from position 129093712 to 129116637 on chromosome 4 (NC_000004.12) and contains 8 exons . Despite its unknown function, it represents an intriguing target for studies in cell biology and cancer research due to its potential roles in fundamental cellular processes .

What are the typical applications for C4orf33 antibodies in research?

C4orf33 antibodies are utilized in multiple experimental techniques to study the expression, localization, and function of this protein. The most common applications include:

• Western Blotting (WB): For detecting C4orf33 protein in cell or tissue lysates, typically showing a band at approximately 23-24 kDa .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of C4orf33 in solution .

• Immunofluorescence (IF): For visualizing the subcellular localization of C4orf33 in fixed cells .

• Immunohistochemistry (IHC): For detecting C4orf33 in paraffin-embedded tissue sections .

Each application requires specific optimization of antibody dilutions and experimental conditions. For instance, recommended dilutions typically range from 1:1000-1:5000 for Western blotting and 1:2000-1:10000 for ELISA applications .

What species reactivity do C4orf33 antibodies typically exhibit?

Most commercially available C4orf33 antibodies demonstrate reactivity to human C4orf33, with many also cross-reacting with mouse and rat orthologs. Some antibodies show broader species reactivity profiles, including:

When selecting an antibody for your research, it's crucial to choose one with validated reactivity to your species of interest. The amino acid sequence conservation between species can affect cross-reactivity, so validation in your specific model system is recommended .

How can I optimize Western blot protocols for C4orf33 detection?

For optimal Western blot detection of C4orf33, consider the following methodological approach:

Sample Preparation:

• Use HEPES lysis buffer (supplemented with protease inhibitors) for cell/tissue lysis .

• For maximum sensitivity, perform ultracentrifugation of lysates (238,700×g for 15 min at 4°C) .

SDS-PAGE and Transfer:

• Use standard SDS-PAGE with appropriate percentage gels (12-15% recommended for this small protein).

• The predicted band size for C4orf33 is 23-24 kDa .

Antibody Incubation:

• Primary antibody: Use at dilutions of 1:1000-1:5000 in appropriate blocking buffer .

• For example, with antibody orb1240825, a dilution of 0.5 μg/mL is recommended .

• Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:10000-1:100000 dilution .

Detection and Validation:

• Include both positive controls (tissues known to express C4orf33 such as mouse kidney, liver, lung, and heart tissues) .

• Include negative controls or knockout samples when available to confirm specificity .

A standard protocol using these parameters has been shown to successfully detect C4orf33 in various tissue samples with high specificity .

What validation methods should I employ to ensure C4orf33 antibody specificity?

Rigorous validation of antibody specificity is crucial for reliable research outcomes. For C4orf33 antibodies, consider implementing this comprehensive validation strategy:

CRISPR/Cas9 Knockout Validation:

• Generate C4orf33 knockout cell lines using CRISPR/Cas9 technology.

• Compare immunoblot results between parental and knockout cells – this is considered the gold standard for validation .

Mosaic Cell Approach:

• Plate wildtype cells expressing a fluorescent marker (e.g., LAMP1-YFP) alongside knockout cells expressing a different marker (e.g., LAMP1-RFP) on the same coverslip.

• This allows direct comparison of antibody staining in genetically identical cells differing only in C4orf33 expression .

Multi-technique Confirmation:

• Validate your antibody using multiple techniques (Western blot, immunofluorescence, immunoprecipitation).

• Concordant results across different methods strengthen confidence in antibody specificity .

Immunoprecipitation-Mass Spectrometry:

• Perform immunoprecipitation with the C4orf33 antibody followed by mass spectrometry.

• This can confirm capture of the target protein and identify potential cross-reactive proteins .

Implementation of these methods can significantly reduce the risk of using non-specific antibodies, which has been identified as a substantial problem in research resulting in questionable findings in highly cited papers .

How do I perform immunofluorescence staining with C4orf33 antibodies?

For successful immunofluorescence detection of C4orf33, follow this optimized protocol based on validated methods:

Cell Preparation:

• Plate cells on glass coverslips and grow to appropriate confluence.

• Fix cells using either:

  • 4% paraformaldehyde for 10 minutes at room temperature, or

  • Chilled methanol (-20°C) for 10 minutes .
    (Note: Compare both fixation methods as protein detection can vary)

Blocking and Permeabilization:

• Block and permeabilize fixed cells in buffer containing TBS, 5% BSA, and 0.3% Triton X-100 (pH 7.4) for 1 hour at room temperature .

Antibody Incubation:

• Primary antibody: Dilute C4orf33 antibody to 2 μg/ml in blocking buffer.

• Incubate cells with primary antibody overnight at 4°C.

• Wash cells 3 times (10 minutes each) with blocking buffer.

• Secondary antibody: Incubate with appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 647) at 1:1000 dilution for 2 hours at room temperature.

• Wash 3 times (10 minutes each) with blocking buffer and once with TBS .

Mounting and Imaging:

• Mount coverslips using fluorescence mounting media.

• Image using confocal microscopy with appropriate laser settings and controls .

This protocol has been successfully used for C4orf33 detection in multiple cell types, including U2OS and HEK-293 cells .

How can computational models help predict antibody specificity for C4orf33?

Advanced computational approaches are revolutionizing antibody design and specificity prediction, which can be applied to C4orf33 research:

Energy Function Modeling:
Recent research has developed computational models that can predict antibody binding profiles by optimizing energy functions associated with different binding modes. These models can be applied to design antibodies with either:

• Cross-specific binding: Allowing interaction with several distinct ligands

• Specific binding: Enabling interaction with only C4orf33 while excluding similar proteins

The process involves:

• Collecting experimental data from phage display selections

• Building computational models based on these datasets

• Optimizing energy functions associated with each binding mode

• Predicting novel antibody sequences with customized specificity profiles

Validation Through Experimental Testing:
These computationally predicted antibody variants can then be experimentally tested to assess the model's capacity to propose novel antibody sequences with customized specificity profiles for C4orf33 .

This approach represents a significant advancement over traditional trial-and-error methods, potentially leading to more specific C4orf33 antibodies for research applications.

What cell types or tissues show significant C4orf33 expression?

Understanding the expression pattern of C4orf33 across different tissues is crucial for contextualizing research findings. The Human Protein Atlas provides comprehensive expression data:

Tissue Expression Profile:
Based on available data, C4orf33 shows differential expression across human tissues . While comprehensive expression data remains limited, experimental evidence from Western blot analyses shows detectable expression in multiple mouse tissues including:

• Kidney

• Liver

• Lung

• Heart

Subcellular Localization:
Though definitive subcellular localization data for C4orf33 remains limited, immunofluorescence studies suggest it may have specific cellular compartmentalization. Further research using knockout-validated antibodies is necessary to conclusively determine its precise localization .

Expression in Disease Models:
Research into C4orf33 expression in disease states is still emerging. The protein's potential roles in cell proliferation and differentiation suggest it could have altered expression in cancer or developmental disorders, making it an intriguing target for further investigation .

How can I use C4orf33 antibodies for protein-protein interaction studies?

Investigating C4orf33 protein interactions can provide critical insights into its function. Here's a methodological approach for protein-protein interaction studies:

Co-Immunoprecipitation Protocol:

• Cell Lysis:

  • Collect cells in HEPES lysis buffer supplemented with protease inhibitors

  • Incubate on ice for 30 minutes

  • Centrifuge at high speed (238,700×g for 15 min at 4°C)

• Pre-clearing:

  • Incubate lysates with empty protein G Sepharose beads for 30 minutes to reduce non-specific binding

• Immunoprecipitation:

  • Couple 1 μg of validated C4orf33 antibody to protein G Sepharose

  • Incubate pre-cleared lysates with antibody-coupled beads for 4-18 hours at 4°C

  • Collect unbound fractions, then wash beads 3-4 times with lysis buffer

• Analysis:

  • Process samples for immunoblotting to detect C4orf33 and potential interacting partners

  • For comprehensive analysis, perform mass spectrometry on immunoprecipitated samples

Mass Spectrometry Approach:
For unbiased identification of C4orf33 interacting proteins:

• Perform immunoprecipitation as described above

• Process samples for trypsin digestion

• Analyze peptides using high-resolution mass spectrometry (e.g., Thermo Orbitrap Fusion)

• Compare results between wildtype and C4orf33 knockout samples to identify specific interactions

This approach has been successfully implemented for other proteins and can reveal novel biological functions through identification of protein complexes.

Why might I see multiple bands in my C4orf33 Western blot?

Multiple bands in C4orf33 Western blots can arise from several factors. Here's a systematic approach to identifying and resolving this issue:

Potential Causes and Solutions:

• Post-translational Modifications:

  • C4orf33 may undergo modifications such as phosphorylation or ubiquitination

  • Solution: Treat samples with phosphatase or deubiquitinating enzymes before SDS-PAGE

• Protein Degradation:

  • Lower molecular weight bands may represent degradation products

  • Solution: Add additional protease inhibitors to lysis buffer and keep samples cold throughout processing

• Alternative Splice Variants:

  • C4orf33 may have uncharacterized splice variants

  • Solution: Validate using C4orf33 knockout controls to determine which bands are specific

• Cross-reactivity:

  • Antibody may bind to other proteins with similar epitopes

  • Solution: Test multiple antibodies targeting different epitopes of C4orf33 and compare results

• Non-specific Binding:

  • Secondary antibody may bind non-specifically to other proteins

  • Solution: Include a secondary-only control and optimize blocking conditions

Expected Band Size:
The predicted molecular weight of C4orf33 is approximately 23-24 kDa . Bands significantly deviating from this size should be carefully evaluated for specificity.

How can I improve signal-to-noise ratio when using C4orf33 antibodies?

Poor signal-to-noise ratio is a common challenge in immunodetection experiments. Here are methodological approaches to improve results with C4orf33 antibodies:

For Western Blotting:

• Optimize Antibody Dilution:

  • Perform a dilution series to find optimal concentration

  • Start with manufacturer recommendations (typically 1:1000-1:5000)

  • For orb1240825 antibody, 0.5 μg/mL has shown good results

• Blocking Optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Extend blocking time to reduce background

• Washing Protocol Enhancement:

  • Increase number of washes (minimum 3×10 minutes)

  • Add low concentrations of detergent (0.05-0.1% Tween-20) to wash buffer

For Immunofluorescence:

• Fixation Method Comparison:

  • Compare paraformaldehyde vs. methanol fixation

  • Different fixation methods can dramatically affect epitope accessibility

• Signal Amplification:

  • Consider using TSA (Tyramide Signal Amplification) systems

  • Use brighter fluorophores (e.g., Alexa Fluor 647 instead of FITC)

• Microscopy Settings:

  • Optimize exposure settings

  • Use spectral unmixing to separate autofluorescence from specific signal

General Recommendations:

• Pre-absorb antibodies with knockout cell lysates to remove cross-reactive antibodies

• Include appropriate positive and negative controls in every experiment

• Consider using FITC or HRP-conjugated C4orf33 antibodies for direct detection

What controls are essential when validating results with C4orf33 antibodies?

Rigorous controls are fundamental to ensuring reliable and reproducible results when working with C4orf33 antibodies. The following controls should be considered essential:

Genetic Controls:

• CRISPR/Cas9 Knockout: The gold standard negative control is a complete knockout of C4orf33. This allows definitive determination of antibody specificity .

• siRNA Knockdown: If knockout is not feasible, siRNA-mediated knockdown can provide partial validation by showing reduced signal intensity.

Technical Controls:

• Primary Antibody Omission: Include samples processed identically but without primary antibody to assess secondary antibody non-specific binding.

• Isotype Control: Use a non-specific antibody of the same isotype (IgG) and concentration to identify potential non-specific binding .

• Peptide Competition: Pre-incubate antibody with the immunizing peptide to confirm epitope-specific binding.

Sample Controls:

• Multiple Tissues/Cell Lines: Test antibody across diverse samples with varying expression levels of C4orf33.

• Overexpression System: Include samples overexpressing tagged C4orf33 to confirm correct molecular weight and localization .

Multi-method Validation:

• Orthogonal Techniques: Confirm findings using multiple methods (e.g., if using Western blot, validate with immunofluorescence) .

• Multiple Antibodies: When possible, use multiple antibodies targeting different epitopes of C4orf33.