CYP4Z1 Antibody

What is CYP4Z1 and why is it significant in cancer research?

CYP4Z1 belongs to the cytochrome P450 superfamily of heme-containing monooxygenases that catalyze the reaction: RH + reduced flavoprotein + O2 = ROH + oxidized flavoprotein + H2O . Unlike most human CYP enzymes that are located on the cytoplasmic side of the endoplasmic reticulum or the matrix side of the inner mitochondrial membrane, CYP4Z1 has been demonstrated on the outer surface of the plasma membrane of cancer cells . This unusual localization has significant implications for cancer research.

CYP4Z1 has garnered research interest due to its differential expression pattern - it is overexpressed in multiple cancer types while showing minimal or no expression in corresponding normal tissues . The enzyme has been consistently linked with poor prognosis and shorter survival times across various cancers:

Furthermore, CYP4Z1 expression is significantly associated with advanced disease stages, high tumor grades, lymph node metastasis, and tumor invasion depth in multiple cancer types . Experimental evidence indicates its involvement in promoting tumor angiogenesis and growth , making it a promising biomarker and potential therapeutic target.

What are the optimal conditions for using CYP4Z1 antibodies in immunohistochemistry?

Successful immunohistochemical detection of CYP4Z1 requires careful optimization of several parameters. Based on multiple published protocols, the following methodology represents current best practices:

Tissue Preparation and Pretreatment

• Use 5μm-thick Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections

• Deparaffinize sections in xylene and rehydrate through a decreasing gradient of alcohols to water

• Block endogenous peroxidase activity with 1-3% hydrogen peroxide for 10-30 minutes at room temperature

• Perform antigen retrieval by microwaving tissues at 650W in 10mM citrate buffer (pH 6.0) for 20 minutes

• Block non-specific binding with 2.5% normal goat serum for 20 minutes at room temperature

Antibody Incubation and Detection

• Primary antibody concentration: Typically 5-10μg/mL, though this varies by manufacturer and should be optimized

• Incubation conditions: Either overnight at 4°C or 1 hour at room temperature depending on the antibody

• Secondary detection: ImmPRESS (Peroxidase) polymer goat anti-rabbit IgG reagent for 30 minutes at room temperature

• Visualization: 3,3'-diaminobenzidine tetrahydrochloride chromogen (DAB) for 2-5 minutes

• Counterstain with Harris's hematoxylin, dehydrate, and mount with coverslips using DPX

Commercial antibody manufacturers recommend varying dilutions; for example, Boster's Anti-CYP4Z1 Antibody (A13386-1) suggests 1:100-1:300 for IHC , while Proteintech recommends optimization for each testing system . Researchers should begin with the manufacturer's recommended dilution and perform titration experiments to determine optimal concentrations for their specific tissue type and fixation conditions.

How should researchers validate the specificity of CYP4Z1 antibodies?

Validating antibody specificity is crucial, particularly for cytochrome P450 family members which share structural similarities. A comprehensive validation approach should include multiple complementary methods:

Blocking Peptide Experiments

Incubate the CYP4Z1 antibody with a specific CYP4Z1 blocking protein (e.g., H00199974-P01, Novus Biologicals) for 60 minutes at room temperature before applying to tissues . Compare staining between sections treated with blocked antibody versus unblocked antibody - the blocking peptide should significantly reduce or eliminate specific staining.

Positive and Negative Controls

• Include known CYP4Z1-positive tissues (e.g., breast cancer tissues) as positive controls

• Use normal tissues with minimal or no CYP4Z1 expression as negative controls

• In one study, strong immunoreactivity was exhibited in positive controls while no observable immunostaining was seen in negative controls

Western Blot Validation

Perform western blot analysis using:

• Recombinant CYP4Z1 protein

• Lysates from cells engineered to express CYP4Z1

• Molecular weight confirmation: CYP4Z1 should appear at approximately 59 kDa

Immunoreactivity Pattern Analysis

Verify that staining is confined to expected cellular locations (membrane and/or cytoplasm) and absent from nuclei . Unexpected staining patterns may indicate cross-reactivity or non-specific binding.

Multiple Antibody Approach

When possible, validate findings using antibodies from different manufacturers or those targeting different epitopes within CYP4Z1.

Multiple studies have employed these validation methods, demonstrating that proper controls can effectively distinguish specific CYP4Z1 staining from background or cross-reactivity with other CYP family members.

What protocols are recommended for Western blot analysis using CYP4Z1 antibodies?

Western blot analysis of CYP4Z1 requires careful optimization to ensure specific detection. Based on published methodologies, the following protocol is recommended:

Sample Preparation

• Culture cells (e.g., CYP4Z1-expressing cell lines) in appropriate conditions

• Consider experimental treatments (e.g., serum starvation, inhibitors) according to research questions

• Extract proteins using standard lysis buffers and determine concentration via Bradford assay

• Prepare 20μg of protein extracts for electrophoresis

Electrophoresis and Transfer

• Separate proteins using 8% polyacrylamide gels

• Transfer to PVDF membrane using standard transfer conditions

• Verify transfer efficiency with reversible protein staining if needed

Antibody Incubation

• Block membrane with 5% nonfat milk in TBST

• Incubate with primary CYP4Z1 antibody at manufacturer-recommended dilution:

  • Proteintech recommends 1:500-1:1000 for their antibody 20142-1-AP

  • Optimize concentration based on signal-to-noise ratio

• Wash thoroughly (typically 3-5 times with TBST)

• Incubate with appropriate HRP-conjugated secondary antibody

Detection and Analysis

• Develop using enhanced chemiluminescence detection

• Expect CYP4Z1 signal at approximately 59 kDa

• Include appropriate loading controls (e.g., β-actin)

For investigators experiencing weak signals or high background, manufacturers often provide troubleshooting guides specific to their antibodies. For example, Proteintech offers a specific Western blot protocol for their CYP4Z1-Specific antibody .

How can epitope mapping be performed for CYP4Z1 antibodies?

Epitope mapping is critical for characterizing antibody binding sites and understanding potential cross-reactivity. The search results describe a high-resolution approach successfully used for mapping anti-CYP4Z1 autoantibody epitopes in breast cancer patients:

PEPperCHIP Microarray Method

• Synthesize an overlapping peptide library comprising 15-mer peptides with an offset of one amino acid, covering the entire CYP4Z1 amino-acid sequence

• Print peptides on standard functionalized glass slides

• Block non-specific binding sites

• Incubate with patient sera (or purified antibodies)

• Incubate with fluorescently labeled secondary antibody

• Subtract signals from secondary antibody-only controls

• Measure relative signal intensity for all overlapping CYP4Z1 peptides

Results Interpretation

In the study examined, this approach successfully identified a peptide (residues 198-212) strongly recognized by all patients' sera, with neighboring peptides also displaying strong signals . Histidine-198 appeared to play a significant role in this epitope.

Structural Context Analysis

Compare identified epitopes with protein structure models (e.g., homology models based on related CYP structures) to determine epitope accessibility. In the reported case, the identified epitope residues (His-198 to Leu-212) were all located on the protein surface, supporting their role as antibody targets .

This epitope mapping approach provides researchers with:

• Precise identification of antibody binding sites

• Information about antibody specificity

• Insights into potential cross-reactivity with related proteins

• Guidance for designing blocking peptides or competitive assays

What are the implications of detecting CYP4Z1 at the cell membrane versus cytoplasm?

The subcellular localization of CYP4Z1 has significant implications for its function and potential as a therapeutic target. Research findings indicate that CYP4Z1 can be detected in both membrane and cytoplasmic compartments:

Membrane Localization

• CYP4Z1 has been demonstrated on the outer surface of the plasma membrane of MCF-7 breast cancer cells by immunofluorescence

• This is unusual for CYP enzymes, which are typically located on the cytoplasmic side of the endoplasmic reticulum or in mitochondria

• Membrane localization may trigger immune responses, leading to the generation of anti-CYP4Z1 autoantibodies in cancer patients

• Surface expression makes CYP4Z1 potentially accessible for antibody-based targeted therapies without requiring cell permeabilization

Cytoplasmic Expression

• CYP4Z1 is also detected in the cytoplasm, which is more consistent with the typical localization of CYP enzymes

• Cytoplasmic expression may reflect the enzyme's role in intracellular metabolic processes

Methodological Considerations

• When scoring CYP4Z1 expression in tissues, researchers consider cells positive if they demonstrate clear membranous and/or cytoplasmic immunoreactivity

• Different antibody clones may show varying affinities for membrane versus cytoplasmic CYP4Z1

• Fixation techniques can affect the detection of membrane versus cytoplasmic staining

Understanding this dual localization pattern is important for developing accurate scoring systems, interpreting staining patterns, and developing potential therapeutic strategies targeting CYP4Z1.

How are anti-CYP4Z1 autoantibodies detected and quantified in cancer patients?

The detection of autoantibodies against CYP4Z1 in patient sera represents a promising approach for cancer diagnosis and monitoring. The following methods have been successfully employed:

Immunoprecipitation Method

• Incubate patient sera with recombinant CYP4Z1 protein and protein A-sepharose beads

• Precipitate resulting immune complexes and wash thoroughly

• Subject to SDS-PAGE and western blot transfer

• Develop western blots using rabbit polyclonal anti-CYP4Z1 antibody and HRP-coupled secondary antibody

• Detect signals using enhanced chemiluminescence

This method demonstrated that "strong signals for CYP4Z1 could be detected in all breast cancer samples while only very weak or no signals were found in control sera" .

Direct Western Blot Analysis

• Perform western blot of recombinant CYP4Z1 protein

• Use patient sera as the primary antibody source

• Use anti-human secondary antibody for detection

• This approach specifically detects antibodies against linear epitopes within CYP4Z1

Indirect ELISA Quantification

• Coat plates with human recombinant CYP4Z1 full-length protein

• Block non-specific binding sites

• Add patient sera or controls at appropriate dilutions

• Incubate with HRP-coupled anti-human secondary antibody

• Measure signals by monitoring HRP-dependent conversion of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) at 415 nm

In one study, all breast cancer sera gave signals between OD415 0.5 and 1.0, while control signals were all significantly lower , demonstrating the quantitative nature of this approach.

These methods can be used complementarily to verify both the presence and quantify the levels of anti-CYP4Z1 autoantibodies in cancer patients, potentially serving as biomarkers for early cancer detection.

How can CYP4Z1 expression be evaluated as a prognostic marker in cancer patients?

Evaluating CYP4Z1 as a prognostic marker requires systematic approaches to scoring, statistical analysis, and clinical correlation. Based on multiple studies, the following methodology is recommended:

Semi-Quantitative Scoring Systems

Most studies employ a 4-point scoring scale:

• Negative (0): Absence of expression or <5% of cells staining

• Low (1): 5-33% of cells showing immunoreactivity

• Intermediate/Moderate (2): 33-66% of cells showing immunoreactivity

• High (3): >67% of cells showing immunoreactivity

For statistical analyses, patients are often grouped into CYP4Z1-positive versus CYP4Z1-negative categories.

Clinical Correlations

Analyze associations between CYP4Z1 expression and:

• Histopathological features (tumor type, grade, stage)

• Clinical parameters (tumor size, invasion depth, lymph node status)

• Patient demographics (age, sex)

Findings from Multiple Studies

CYP4Z1 has been identified as an independent predictor of poor prognosis in several cancer types:

• In cervical cancer, CYP4Z1 expression was significantly correlated with shorter survival times (78% survival rate in CYP4Z1-positive patients vs. 86.7% in CYP4Z1-negative patients)

• In ovarian cancer, CYP4Z1 expression was "correlated with shorter patient survival and has been identified as an independent indicator of a poor prognosis"

• In triple-negative breast cancer, CYP4Z1 expression "was correlated with the survival of TNBC patients" and "was an independent determinant of the poor prognosis of TNBC"

These consistent findings across multiple cancer types suggest CYP4Z1 expression has robust prognostic value.

What methodological approaches can distinguish CYP4Z1 from other CYP family members?

Distinguishing CYP4Z1 from other cytochrome P450 family members is crucial for accurate research outcomes. Several complementary approaches can be employed:

Antibody Selection and Validation

• Choose antibodies raised against unique regions of CYP4Z1

• For example, Boster's antibody (A13386-1) was produced against a synthesized peptide derived from human CYP4Z1 (AA range: 71-120)

• Proteintech's antibody (20142-1-AP) is designated as "CYP4Z1-Specific"

• Validate antibody specificity against recombinant CYP4Z1 and closely related CYP family members via Western blot

Blocking Peptide Controls

• Incubate primary antibody with a CYP4Z1-specific blocking peptide

• Apply this mixture to tissues in place of the primary antibody alone

• Compare staining density between blocked and unblocked antibody samples

• This approach confirmed antibody specificity in multiple studies

Multiple Detection Methods

• Combine protein detection (Western blot, IHC) with mRNA analysis (qPCR, in situ hybridization)

• CYP family members have distinct mRNA sequences that can be more easily differentiated than proteins

• Correlation between protein and mRNA detection provides stronger evidence of specificity

Engineered Cell Controls

• Use cell lines engineered to specifically express CYP4Z1

• Include cells expressing related CYP family members as controls

• This approach was successfully used to confirm antibody specificity

Mass Spectrometry Validation

• For the most definitive identification, consider proteomics approaches

• Immunoprecipitate the target protein and confirm identity via mass spectrometry

• This can unequivocally distinguish between closely related CYP family members

By combining these approaches, researchers can ensure their findings specifically relate to CYP4Z1 rather than other members of the cytochrome P450 family.

What is the relationship between CYP4Z1 expression and tumor angiogenesis/growth?

Experimental evidence has established connections between CYP4Z1 expression and tumor progression through multiple mechanisms:

Direct Experimental Evidence

Research has specifically investigated "the effects of increased CYP4Z1 expression on tumor angiogenesis and growth of human mammary carcinomas in vitro and in vivo" . These studies compared CYP4Z1-expressing cells (T47D-CYP4Z1) to vector control cells, providing direct evidence of CYP4Z1's role in cancer progression.

Signaling Pathway Involvement

CYP4Z1 appears to influence tumor growth and angiogenesis through specific signaling pathways:

• The MAPK and PI3K/Akt signaling pathways are involved in CYP4Z1-mediated regulation of angiogenic molecules

• Experimental approaches used specific inhibitors:

  • Wortmannin for PI3K inhibition

  • SP600125 for JNK inhibition

  • SB203580 for p38 MAPK inhibition

  • U0126 for ERK inhibition

• Additional validation employed siRNA against ERK1/2 or Akt

Angiogenic Factor Production

CYP4Z1 expression influences the production of angiogenic factors:

• Levels of angiogenic factors in cell culture supernatants can be quantified using ELISA kits

• These factors promote the formation of new blood vessels to support tumor growth

Clinical Correlations

Multiple clinical studies support CYP4Z1's role in tumor progression:

• CYP4Z1 expression correlates with advanced disease stages and high tumor grades across multiple cancer types

• Significant associations exist between CYP4Z1 expression and:

  • Lymph node metastasis

  • Tumor invasion depth

  • Poor patient survival

These findings collectively suggest that CYP4Z1 promotes cancer progression by stimulating angiogenesis through specific signaling pathways, making it a potential therapeutic target for cancer treatment.