CYP98A8 Antibody

What is CYP2C8 and what is its role in human metabolism?

CYP2C8 is a cytochrome P450 monooxygenase that plays crucial roles in metabolizing various endogenous substrates, including fatty acids, steroid hormones, and vitamins. Mechanistically, it functions by inserting one oxygen atom into a substrate while reducing the second oxygen atom into a water molecule, with electrons provided by NADPH via cytochrome P450 reductase. CYP2C8 primarily catalyzes the epoxidation of double bonds in polyunsaturated fatty acids (PUFA), with a preference for the last double bond, and also catalyzes carbon-hydrogen bond hydroxylation .

Beyond its role in fatty acid metabolism, CYP2C8:

• Metabolizes all-trans retinoic acid to its 4-hydroxylated form

• Displays 16-alpha hydroxylase activity toward estrogen steroid hormones

• Serves as the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (taxol)

What types of CYP2C8 antibodies are currently available for research applications?

Several types of CYP2C8 antibodies are available for research applications:

Most commercially available antibodies are provided as unconjugated primary antibodies in buffered aqueous glycerol solutions and have undergone validation for specific applications .

What tissue samples are optimal for validating CYP2C8 antibodies?

For optimal validation of CYP2C8 antibodies, liver tissue represents the gold standard as it expresses the highest levels of CYP2C8. According to research data, human liver shows abundant expression, while mouse and rat liver tissues also demonstrate cross-reactivity with many CYP2C8 antibodies, making them suitable validation models .

Beyond the liver, CYP2C8 mRNA has been detected in kidney, testes, adrenal gland, prostate, brain, uterus, mammary gland, ovary, lung, and duodenum, though at substantially lower levels . When validating antibody performance in these extrahepatic tissues, expect significantly weaker signals that may require optimized detection methods .

How can I evaluate the specificity of CYP2C8 antibodies against other CYP2C family members?

Evaluating antibody specificity against other CYP2C family members requires a multi-faceted approach due to the high sequence homology between CYP2C8, CYP2C9, CYP2C18, and CYP2C19.

Methodological approach:

• Sequence Analysis: Compare the immunogen sequence used to generate the antibody with sequences of other CYP2C family members. For example, the immunogen sequence for Sigma's HPA013970 antibody is documented, allowing for BLAST analysis to identify potential cross-reactive regions .

• Recombinant Protein Testing: Test the antibody against purified recombinant proteins of all CYP2C family members under identical conditions.

• Orthogonal Validation: Utilize orthogonal RNAseq validation, as implemented for the HPA013970 antibody, to confirm correlation between detected protein and mRNA expression patterns .

• Genetic Knockdown Controls: Employ siRNA or CRISPR-based knockdown/knockout models specific to each CYP2C family member to evaluate antibody cross-reactivity.

• Tissue Panel Testing: Some antibodies undergo validation against extensive tissue arrays (44 normal human tissues and 20 common cancer types) and protein arrays (364 human recombinant protein fragments) to assess specificity .

What are the optimal conditions for using CYP2C8 antibodies in immunohistochemistry applications?

Successful immunohistochemistry (IHC) with CYP2C8 antibodies requires careful optimization of several parameters:

Sample Preparation:

• For formalin-fixed paraffin-embedded (FFPE) tissues, proper fixation time (24-48 hours) is critical

• Antigen retrieval is essential - for Proteintech's 16546-1-AP antibody, TE buffer at pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

Protocol Optimization:

• Dilution ranges vary by antibody: HPA013970 is recommended at 1:50-1:200 for IHC

• Incubation time: typically overnight at 4°C for optimal results

• Detection system: DAB chromogen provides good sensitivity and permanence

Control Samples:

• Positive control: human liver tissue (primary site of CYP2C8 expression)

• Negative control: tissues known not to express CYP2C8

• Antibody controls: sections processed without primary antibody

Signal Interpretation:

• CYP2C8 should display predominantly endoplasmic reticulum localization

• Positive staining in liver shows characteristic hepatocyte distribution

• When evaluating IHC results in non-liver tissues, expect significantly weaker signals

How can computational approaches improve the design of CYP2C8-specific antibodies?

Recent advances in computational antibody design offer promising approaches for developing CYP2C8-specific antibodies:

Computational design methodology:

• Phage Display Selection: Initial phage-display experiments with antibody libraries are performed against CYP2C8 and related proteins. This generates training datasets of sequences with various binding profiles .

• Binding Mode Identification: Computational models identify different binding modes associated with particular ligands (like CYP2C8) against which antibodies are either selected or not selected .

• Energy Function Optimization: For designing specific antibodies, researchers minimize the energy functions associated with desired targets (CYP2C8) while maximizing those for undesired targets (other CYP2C family members) .

• Model Validation: The computational approach is experimentally validated by synthesizing and testing antibodies with predicted specificity profiles .

The power of this approach lies in its ability to disentangle binding modes even when associated with chemically very similar ligands, such as closely related CYP2C family members. This enables the design of antibodies with customized specificity profiles, either with high affinity for a particular target or with cross-specificity for multiple targets .

What are the best practices for western blot optimization with CYP2C8 antibodies?

Optimizing western blot protocols for CYP2C8 requires attention to several factors:

Sample Preparation:

• Microsomal preparation is recommended for enrichment of ER-localized CYP2C8

• Include protease inhibitor cocktails to prevent degradation

• For liver tissue, gentle lysis buffers preserve membrane protein integrity

Antibody Selection and Dilution:

• For Sigma's HPA013970: use 0.04-0.4 μg/mL for immunoblotting

• For Abcam's ab88904: dilution range of 1:2000-1:10000 is recommended

• For Proteintech's 16546-1-AP: dilution range of 1:2000-1:10000 is optimal

Blocking and Membrane Conditions:

• 5% non-fat dry milk or BSA in TBST provides effective blocking

• PVDF membranes generally offer better results for membrane proteins like CYP2C8

• Transfer conditions: 100V for 1 hour or 30V overnight at 4°C for complete transfer

Detection and Analysis:

• HRP-conjugated secondary antibodies with enhanced chemiluminescence provide good sensitivity

• Expected molecular weight for CYP2C8 is approximately 56 kDa

• Positive controls should include human liver tissue, while mouse and rat liver are appropriate for cross-reactivity studies

How do genetic polymorphisms in CYP2C8 affect antibody binding and experimental interpretation?

Genetic polymorphisms in CYP2C8 can significantly impact antibody binding and experimental interpretation:

Common polymorphisms and their effects:

• Several polymorphisms in CYP2C8 are associated with decreased metabolism of paclitaxel and arachidonic acid

• Bisphosphonate-related osteonecrosis of the jaw is associated with polymorphisms of CYP2C8 in multiple myeloma patients

Impact on antibody-based detection:

• Epitope Alterations: Polymorphisms occurring within the epitope recognized by an antibody may reduce binding affinity or completely prevent recognition.

• Expression Level Variations: Some polymorphisms affect protein expression levels rather than sequence, potentially leading to variable signal intensity that may be misinterpreted as technical variation.

• Solution Strategies:

  • Use antibodies targeting conserved regions less likely to contain polymorphic sites

  • Employ multiple antibodies targeting different epitopes

  • Consider genotyping samples when inconsistent results are observed

  • Correlate protein detection with enzymatic activity assays for functional validation

What are the optimal storage and handling conditions for maintaining CYP2C8 antibody activity?

To maintain optimal CYP2C8 antibody activity, follow these storage and handling recommendations:

Storage Conditions:

• Store at -20°C for long-term preservation (as specified for HPA013970)

• Many CYP2C8 antibodies are provided in buffered aqueous glycerol solutions that act as cryoprotectants

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

Handling Protocols:

• When removing from freezer storage, thaw antibodies on ice

• Centrifuge briefly after thawing to collect liquid at the bottom of the tube

• Wear gloves to prevent contamination with proteins from skin

• For shipping purposes, wet ice is typically used (as noted for HPA013970)

Working Solution Preparation:

• Prepare working dilutions in appropriate buffers just before use

• For western blotting, typically dilute in blocking buffer

• For IHC, dilute in manufacturer-recommended antibody diluent

• Return stock solutions to -20°C immediately after use

How should I interpret inconsistent CYP2C8 antibody results across different experimental platforms?

When facing inconsistent results across different experimental platforms, systematic analysis is required:

Platform-specific considerations:

• Western Blot vs. IHC Discrepancies:

  • Epitope accessibility differs between platforms - denatured proteins in WB vs. partially preserved structure in IHC

  • Certain antibodies perform better in specific applications (e.g., HPA013970 has validated protocols for both WB and IHC)

• Quantitative vs. Qualitative Discrepancies:

  • Western blot provides semi-quantitative data while IHC is primarily qualitative

  • Consider whether discrepancies relate to detection sensitivity or specificity

Methodological approach to resolving inconsistencies:

• Review antibody validation data - some antibodies undergo extensive validation including orthogonal RNAseq validation and protein array testing

• Implement technical controls:

  • Use recombinant CYP2C8 protein as a standard

  • Include tissues with known expression (liver) as positive controls

  • Test multiple antibody dilutions to establish optimal signal-to-noise ratio

• Correlate with orthogonal methods:

  • RT-qPCR for mRNA expression

  • Enzymatic activity assays

  • Mass spectrometry-based protein identification

What controls are essential when designing experiments with CYP2C8 antibodies?

A comprehensive control strategy is essential for reliable experiments with CYP2C8 antibodies:

Essential controls by application:

• For Western Blot:

  • Positive tissue controls: Human liver tissue (primary expression site), mouse or rat liver (cross-reactivity confirmed for many antibodies)

  • Loading controls: β-actin, GAPDH, or other housekeeping proteins

  • Negative controls: Tissues not expressing CYP2C8

  • Technical controls: Secondary antibody only, gradient of recombinant protein

• For Immunohistochemistry:

  • Tissue controls: Human liver cancer tissue and normal liver tissue

  • Technical controls: No primary antibody, isotype control

  • Antigen retrieval controls: Different pH buffers (TE buffer pH 9.0 recommended for some antibodies)

• For All Applications:

  • Antibody titration: Test multiple dilutions to establish optimal signal-to-noise ratio

  • Specificity controls: Pre-absorption with immunogen peptide when available

  • Genetic controls: siRNA knockdown or CRISPR knockout samples when possible