CYP2C18 Antibody

What is CYP2C18 and what is its function in human metabolism?

CYP2C18 is a member of the cytochrome P450 family of enzymes involved in drug metabolism. It has been characterized at the molecular level, though the protein has not been extensively purified from liver tissue. CYP2C18 metabolizes several compounds including tolbutamide, (S)-mephenytoin, warfarin, tienilic acid, diclofenac, aminopyrine, and bisphenol A . The enzyme catalyzes the oxidation of tolbutamide to hydroxytolbutamide, which can be measured as an indicator of its activity . While CYP2C18 is expressed at very low levels in human liver, it appears to be a major CYP2C enzyme in the skin and lungs, with mRNA also detected in the brain, uterus, mammary gland, kidney, and duodenum .

What types of CYP2C18 antibodies are currently available for research?

Several types of CYP2C18 antibodies are available for research applications:

• By reactivity: Antibodies specific to human CYP2C18, as well as those cross-reactive with mouse and rat CYP2C18

• By host: Most commonly rabbit-derived polyclonal antibodies

• By application: Antibodies validated for Western Blot (WB), Immunohistochemistry (IHC), Immunohistochemistry-paraffin (IHC-p), and Enzyme-Linked Immunosorbent Assay (ELISA)

• By clonality: Primarily polyclonal antibodies, with specific clones like RB16998 also available

How does CYP2C18 differ from other members of the CYP2C family?

While the search results don't explicitly compare CYP2C18 to other CYP2C family members, we know that CYP2C18 has distinct tissue expression patterns compared to other family members. Unlike some other CYP2C enzymes that are abundantly expressed in the liver, CYP2C18 is expressed at very low levels in human liver but has notable expression in extrahepatic tissues like skin and lung . Additionally, CYP2C18 has specific substrate preferences and metabolizes compounds like tolbutamide with measurable hydroxylase activity (0.509 ± 0.052 μmol·min⁻¹·g⁻¹ S9 protein or 8.82 ± 0.90 mol·min⁻¹·mol⁻¹ CYP) . A spliced variant of CYP2C18 with exon 5 missing has also been identified, which may have distinct functional implications .

What are the validated applications for CYP2C18 antibodies?

CYP2C18 antibodies have been validated for several experimental applications:

• Western Blotting (WB): For detecting CYP2C18 protein in tissue or cell lysates

• Immunohistochemistry (IHC): For localizing CYP2C18 in tissue sections

• IHC-paraffin (IHC-p): For detecting CYP2C18 in paraffin-embedded tissues

• ELISA: For quantitative detection of CYP2C18

Most commercially available antibodies specify the dilutions recommended for each application. For example, some vendors recommend dilutions of 1/500 - 1/2000 for Western blotting and 1 μg/ml for ELISA .

How should CYP2C18 antibodies be stored and handled to maintain their activity?

Based on standard antibody handling practices and the information provided in the search results, CYP2C18 antibodies should be:

• Stored as aliquots at -20°C to avoid repeated freeze/thaw cycles which can degrade antibody quality

• Typically provided in a buffer containing PBS (pH 7.3), 0.02% sodium azide, and 50% glycerol

• Used at the recommended dilutions for specific applications (e.g., 1/500 - 1/2000 for WB, 1 μg/ml for ELISA)

• Allowed to reach room temperature before use, but kept cold during storage

What is the expected molecular weight of CYP2C18 in Western blot experiments?

The calculated molecular weight of CYP2C18 is approximately 56 kDa, but the observed molecular weight in Western blot experiments is typically around 50 kDa . This difference between calculated and observed molecular weights is not uncommon for proteins and can be due to various factors including post-translational modifications, protein folding, or the specific gel system used for separation.

How can CYP2C18 enzymatic activity be measured in experimental systems?

The enzymatic activity of CYP2C18 can be measured by its ability to catalyze the oxidation of tolbutamide to hydroxytolbutamide. In research settings, this can be quantified using High-Performance Liquid Chromatography (HPLC) . In a study establishing a transgenic cell line expressing CYP2C18, researchers measured tolbutamide hydroxylase activity in the postmitochondrial supernatant (S9) fraction of cells. The specific activity was determined to be 0.509 ± 0.052 μmol·min⁻¹·g⁻¹ S9 protein or 8.82 ± 0.90 mol·min⁻¹·mol⁻¹ CYP . This assay provides a functional readout of CYP2C18 activity that complements the detection of protein expression using antibodies.

What are the implications of the CYP2C18 splice variant missing exon 5?

A CYP2C18 cDNA clone with exon 5 missing has been identified during cloning experiments . Exon 5 is believed to be necessary for increasing electron transfer for certain CYP forms and specific substrates . Therefore, this splice variant may have altered electron transfer capabilities, potentially affecting enzyme activity and substrate specificity. The functional consequences of this splice variant warrant further investigation, as it could represent a naturally occurring isoform with distinct metabolic properties. Researchers studying CYP2C18 should be aware of this variant and consider its potential implications for their experimental systems and interpretations.

How can CYP2C18 expression and activity be modulated in experimental systems?

Unlike some other CYP enzymes, CYP2C18 mRNA cannot be induced by typical CYP inducers such as rifampicin or phenobarbital in human livers or cultured primary hepatocytes . This presents a challenge for researchers seeking to study CYP2C18 function. One approach to overcome the low endogenous expression is to establish transgenic cell lines expressing recombinant CYP2C18, as demonstrated by the creation of the CHL-CYP2C18 cell line . This cell line was developed by:

• Cloning CYP2C18 cDNA from human liver using RT-PCR

• Subcloning into a mammalian expression vector (pREP9)

• Transfecting Chinese Hamster Lung (CHL) cells, which have limited endogenous CYP enzyme activities but adequate levels of supporting proteins like NADPH-cytochrome P450 oxidoreductase (OR) and cytochrome b5

Such engineered systems provide a controlled environment for studying CYP2C18 function independent of its natural low expression levels.

How can researchers validate the specificity of CYP2C18 antibodies?

To ensure the specificity of CYP2C18 antibodies, researchers should:

• Include positive controls: Use samples known to express CYP2C18 (e.g., CHL-CYP2C18 cell line)

• Include negative controls: Test the antibody on samples known not to express CYP2C18 (e.g., parental CHL cells)

• Confirm the expected molecular weight: Verify that the detected band appears at the expected molecular weight (~50 kDa observed)

• Cross-validation: Use multiple antibodies targeting different epitopes of CYP2C18 if available

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm signal specificity

• Knockdown validation: If possible, test the antibody in CYP2C18 knockdown or knockout systems

What are the key considerations when designing experiments to study CYP2C18 expression in tissues?

When studying CYP2C18 expression in tissues, researchers should consider:

• Tissue selection: Focus on tissues known to express CYP2C18, including skin, lung, brain, uterus, mammary gland, kidney, and duodenum, rather than just liver where expression is low

• Detection method sensitivity: Choose methods with appropriate sensitivity given the generally low expression levels of CYP2C18

• Species differences: Be aware that antibody reactivity may vary between species (human, mouse, rat)

• Antibody selection: Choose antibodies validated for the specific application (WB, IHC, ELISA) and tissue of interest

• Fixation and processing: For IHC applications, consider how tissue fixation and processing might affect epitope accessibility

• Quantification approach: For relative or absolute quantification, establish appropriate normalization controls

How do researchers distinguish between CYP2C18 and other closely related CYP2C family members?

Distinguishing between CYP2C18 and other CYP2C family members (such as CYP2C8, CYP2C9, and CYP2C19) can be challenging due to high sequence homology. Researchers should:

• Use highly specific antibodies: Select antibodies that have been validated for minimal cross-reactivity with other CYP2C family members

• Target unique epitopes: Choose antibodies that target regions of CYP2C18 that differ from other family members

• Functional assays: Complement antibody-based detection with functional assays that exploit substrate specificity differences between CYP2C enzymes

• Recombinant standards: Include recombinant CYP2C18 and other CYP2C proteins as standards to assess antibody specificity

• Mass spectrometry: For definitive identification, consider peptide mass fingerprinting or other mass spectrometry approaches that can distinguish between closely related proteins

What cell lines are suitable for studying CYP2C18 function?

Given the low endogenous expression of CYP2C18 in many cell types, engineered cell lines are particularly valuable for studying this enzyme. The established CHL-CYP2C18 cell line provides an effective model system . Chinese Hamster Lung (CHL) cells were selected as the host cell line because they have limited endogenous CYP enzyme activities but contain adequate levels of supporting proteins like NADPH-cytochrome P450 oxidoreductase (OR) and cytochrome b5 to support CYP activities . The pREP9 expression vector used for establishing this cell line contains the Epstein-Barr virus (EBV) origin of replication and nuclear antigen (EBNA-1), allowing high-copy episomal replication in mammalian cells . The Rous sarcoma virus long terminal repeat (RSV LTR) early promoter controls the expression of the CYP2C18 cDNA in this system .

How might CYP2C18 research contribute to personalized medicine?

CYP2C18's role in metabolizing various drugs including tolbutamide, (S)-mephenytoin, warfarin, tienilic acid, and diclofenac suggests it may contribute to inter-individual differences in drug responses. While the search results don't specifically address pharmacogenomics of CYP2C18, research into genetic variants, splice variants (like the exon 5 deletion variant) , and tissue-specific expression patterns could potentially inform personalized dosing strategies for medications metabolized by this enzyme. The development of specific antibodies and functional assays for CYP2C18 facilitates such research by enabling the detection and quantification of this enzyme in clinical samples.

What novel technological approaches are emerging for studying CYP2C18?

While the search results don't explicitly describe emerging technologies for CYP2C18 research, several approaches could advance this field:

• CRISPR/Cas9 gene editing: For creating precise knockout or knockin models of CYP2C18

• Single-cell analysis: To understand cell-type specific expression patterns within heterogeneous tissues

• Organoid systems: For studying CYP2C18 function in more physiologically relevant 3D tissue models

• Computational modeling: To predict substrate binding and metabolism by CYP2C18

• Antibody engineering: Development of more specific antibodies or nanobodies targeting CYP2C18