CYTB5-B Antibody

What is CYTB5B and what are its key structural characteristics?

Cytochrome b5 type B (also known as OMB5 or CYB5-M) is a 146 amino acid membrane-bound hemoprotein that acts as an electron carrier for several membrane-bound oxygenases . It contains one cytochrome b5 heme-binding domain and is encoded by a gene that maps to human chromosome 16q22.1 . The protein has a calculated molecular weight of 16 kDa but is typically observed at 20-21 kDa in experimental conditions, likely due to post-translational modifications . Both cytochrome b5 (cyt b5) and cytochrome b5 reductase (b5R) have a cytosolic domain containing cofactors required for electron transfer and a single transmembrane (TM) helix that serves not only as a membrane anchor but also plays a functional role in protein-protein interactions .

How does CYTB5B differ from other cytochrome b5 family members?

Cytochrome b5 type B is specifically localized to the outer mitochondrial membrane, distinguishing it from other family members . While all cytochrome b5 proteins function as electron carriers, CYTB5B has unique expression patterns and protein interactions. Unlike other cytochrome proteins, CYTB5B is expressed on the plasma membrane of lymphoma cells but not normal lymphocytes, reactive lymphocytes, or bone marrow precursor cells, suggesting potential diagnostic applications in oncology . The transmembrane helices of CYTB5B mediate the formation of stable complexes with proteins like cytochrome b5 reductase (b5R) and stearoyl-CoA desaturase-1 (SCD1), highlighting its role in specific metabolic pathways .

What criteria should be considered when selecting a CYTB5B antibody for research?

When selecting a CYTB5B antibody, researchers should consider:

• Antibody type: Both monoclonal (e.g., mouse IgG1 κ) and polyclonal (e.g., rabbit IgG) CYTB5B antibodies are available with distinct advantages .

• Species reactivity: Confirm cross-reactivity with your species of interest. Many CYTB5B antibodies react with human, mouse, and rat samples .

• Application compatibility: Verify validation for your intended applications (WB, IP, IF, IHC-P, ELISA) .

• Epitope recognition: Consider whether the antibody recognizes specific domains or regions of the protein based on the immunogen information .

• Validation data: Review published literature and manufacturer validation data showing successful application in experimental contexts similar to yours .

The selection should be guided by your specific experimental requirements and the quality of validation data available.

How can I validate the specificity of a CYTB5B antibody?

To validate CYTB5B antibody specificity:

• Positive and negative controls: Use tissues/cells known to express or lack CYTB5B. Liver tissues and HepG2 cells show high expression and serve as excellent positive controls .

• Knockdown/knockout validation: Compare antibody reactivity in wild-type versus CYTB5B knockdown/knockout samples .

• Western blot analysis: Confirm the detection of the expected band size (approximately 20-21 kDa) .

• Peptide competition assay: Pre-incubation with the immunizing peptide should block specific binding.

• Multiple antibody approach: Use at least two antibodies raised against different epitopes to confirm consistent detection patterns.

For example, published data shows successful detection of CYTB5B in HepG2 cells, mouse liver tissue, human liver tissue, mouse lung tissue, human adrenal gland tissue, rat liver tissue, and rat lung tissue by Western blot , providing excellent benchmark samples for validation.

What are the optimal conditions for Western blot detection of CYTB5B?

For optimal Western blot detection of CYTB5B:

• Sample preparation: Use tissue or cell lysates with appropriate detergents to solubilize membrane proteins. HepG2, liver tissues, and lung tissues are excellent sources of CYTB5B .

• Antibody dilution: Use recommended dilutions (typically 1:2000-1:10000 for polyclonal antibodies or 1:500-1:2000 ).

• Blocking conditions: 5% non-fat dry milk or BSA in TBST is generally effective.

• Expected band size: Look for bands at 20-21 kDa, though the calculated weight is 16 kDa .

• Sample loading: Load 20-50 μg of total protein per lane.

Representative Western blot results from multiple tissue and cell line samples demonstrate the expected banding pattern, with strong signals observed in liver tissues and HepG2 cells .

What protocols are recommended for immunohistochemical detection of CYTB5B?

For immunohistochemical detection of CYTB5B:

• Tissue preparation: Formalin-fixed, paraffin-embedded sections work well for CYTB5B detection .

• Antigen retrieval: TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be effective .

• Antibody dilution: Use dilutions of 1:20-1:200 for optimal staining .

• Detection system: Both chromogenic and fluorescent detection systems are compatible with CYTB5B antibodies.

• Positive control tissues: Human and mouse testis tissues have been validated for CYTB5B immunohistochemistry .

Proper controls include omission of primary antibody and use of tissues with known CYTB5B expression patterns. Counterstaining with DAPI for nuclear visualization is recommended for immunofluorescence approaches.

How can CYTB5B antibodies be used in immunoprecipitation experiments?

For immunoprecipitation of CYTB5B:

• Lysate preparation: Use 1.0-3.0 mg of total protein lysate with detergents suitable for membrane proteins.

• Antibody amount: Use 0.5-4.0 μg of antibody per immunoprecipitation reaction .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Incubation conditions: Incubate antibody with lysate overnight at 4°C with gentle rotation.

• Washing conditions: Use stringent washing to reduce background while preserving specific interactions.

This approach is particularly valuable for studying CYTB5B interactions with partners like cytochrome b5 reductase (b5R) and stearoyl-CoA desaturase-1 (SCD1), which form stable binary and ternary complexes .

What are common challenges when detecting CYTB5B and how can they be overcome?

Common challenges and solutions include:

• Membrane protein solubilization: CYTB5B is a membrane-bound protein that may be difficult to extract.

  • Solution: Use stronger detergents like SDS (for WB) or NP-40/Triton X-100 (for IP) to improve solubilization.

• Non-specific binding: Multiple bands in Western blot.

  • Solution: Increase blocking time/concentration, optimize antibody dilution, and use more stringent washing conditions.

• Weak signal in tissue sections:

  • Solution: Optimize antigen retrieval methods, use signal amplification systems, and consider longer primary antibody incubation times.

• Inconsistent results between applications:

  • Solution: Verify antibody validation for each specific application and optimize protocols accordingly. Some antibodies work well for WB but poorly for IHC or IF .

• Variability in expression levels:

  • Solution: Include appropriate controls and normalize to housekeeping proteins when quantifying expression.

How should researchers optimize CYTB5B immunofluorescence protocols?

For optimal CYTB5B immunofluorescence:

• Fixation method: 4% paraformaldehyde typically preserves CYTB5B antigenicity well.

• Permeabilization: Use 0.1-0.3% Triton X-100 or 0.1% saponin to allow antibody access to membrane proteins.

• Antibody dilution: Start with 1:50-1:500 dilutions and optimize based on signal-to-noise ratio .

• Blocking: 5-10% normal serum from the species of the secondary antibody helps reduce background.

• Co-localization markers: Include markers for mitochondria (e.g., MitoTracker) or endoplasmic reticulum to confirm proper subcellular localization.

HepG2 cells have been validated for immunofluorescence detection of CYTB5B and serve as an excellent positive control .

How can CYTB5B antibodies be used to study protein-protein interactions in electron transport systems?

CYTB5B participates in critical protein-protein interactions within electron transport systems:

• Co-immunoprecipitation: Use CYTB5B antibodies to pull down complexes containing cytochrome b5 reductase (b5R) and stearoyl-CoA desaturase-1 (SCD1) .

• Proximity ligation assay: Detect in situ interactions between CYTB5B and binding partners with high sensitivity.

• FRET/BRET analysis: Combine CYTB5B antibodies with fluorescently tagged partner proteins to study interaction dynamics.

• Crosslinking studies: Use membrane-permeable crosslinkers to stabilize transient interactions before immunoprecipitation.

• Deletion mutant analysis: Combine with transmembrane domain deletion constructs to assess the role of TM helices in complex formation .

Recent research demonstrates that CYTB5B forms stable binary complexes with b5R and SCD1, and these proteins can assemble into a stable ternary complex . The transmembrane helices are crucial for these interactions, suggesting they serve functions beyond mere membrane anchoring.

What is the significance of CYTB5B in cancer research and how can antibodies contribute to this field?

CYTB5B shows promising relevance in cancer research:

• Differential expression analysis: CYTB5B is expressed on the plasma membrane of lymphoma cells but not normal lymphocytes, reactive lymphocytes, or bone marrow precursor cells .

• Tissue microarray analysis: Use CYTB5B antibodies to screen expression patterns across tumor types and correlate with clinical outcomes.

• Flow cytometry applications: Develop protocols using conjugated CYTB5B antibodies for detection in hematological malignancies.

• Metabolic profiling: Investigate CYTB5B's role in altered lipid metabolism in cancer cells through antibody-based detection of protein complexes.

• Therapeutic target validation: Combine with siRNA knockdown or CRISPR knockout studies to assess CYTB5B as a potential therapeutic target.

The selective expression of CYTB5B in lymphoma cells suggests potential diagnostic applications and warrants further investigation into its role in cancer metabolism and proliferation.

How can researchers integrate CYTB5B antibody-based techniques with high-throughput or systems biology approaches?

Integration of CYTB5B antibody techniques with systems approaches:

• Proteomics workflow:

  • Use CYTB5B antibodies for immunoprecipitation followed by mass spectrometry to identify novel interaction partners.

  • Compare interactomes across different cell types or disease states.

• Multi-omics integration:

  • Correlate CYTB5B protein expression data with transcriptomics and metabolomics datasets to elucidate its role in cellular pathways.

  • Develop predictive models of electron transport efficiency based on protein complex formation.

• High-content imaging:

  • Utilize CYTB5B antibodies in automated imaging platforms to screen for factors affecting subcellular localization or expression levels.

  • Combine with other markers to create multiplexed imaging panels for studying metabolic processes.

• Single-cell analysis:

  • Adapt CYTB5B antibody protocols for single-cell Western blotting or imaging mass cytometry to examine cell-to-cell variability in expression.

This integrated approach provides a more comprehensive understanding of CYTB5B's role within the complex cellular machinery of electron transport and lipid metabolism.

What is known about the role of CYTB5B transmembrane domains in protein complex formation?

Recent research has revealed that the transmembrane (TM) helices of CYTB5B play crucial functional roles beyond membrane anchoring:

• Complex formation: The TM helices mediate the formation of stable binary complexes between CYTB5B and cytochrome b5 reductase (b5R), as well as between CYTB5B and stearoyl-CoA desaturase-1 (SCD1) .

• Ternary complex assembly: CYTB5B, b5R, and SCD1 can form a stable ternary complex, with the TM helices being essential for this assembly .

• Structural requirements: Deletion or mutation studies of the TM domains provide insight into the specific amino acid sequences required for these interactions.

• Membrane microdomains: The TM domains may localize CYTB5B to specific membrane microdomains, facilitating efficient electron transfer.

• Conformational changes: The TM helix could influence conformational changes in the cytosolic domain that affect electron transfer efficiency.

Understanding these molecular interactions has significant implications for metabolic pathways involving electron transfer and lipid metabolism.

How can CYTB5B antibodies contribute to understanding mitochondrial dysfunction in disease?

CYTB5B antibodies can advance research on mitochondrial dysfunction:

• Biomarker development: Assess changes in CYTB5B expression or localization as potential biomarkers for mitochondrial disorders.

• Pathological samples analysis: Compare CYTB5B distribution and complex formation in healthy versus diseased tissues.

• Drug screening: Use CYTB5B antibodies to evaluate the effects of therapeutic compounds on restoring normal protein interactions.

• Aging research: Study changes in CYTB5B expression and function during cellular senescence and aging-related mitochondrial decline.

• Neurodegenerative disorders: Investigate CYTB5B in diseases with known mitochondrial involvement, such as Parkinson's and Alzheimer's.

Since CYTB5B is localized to the outer mitochondrial membrane and participates in electron transfer processes, it represents an important target for understanding disruptions in cellular energetics and redox homeostasis that underlie many diseases.