UGT2B10 Antibody

What is UGT2B10 and why is it important in research?

UGT2B10 is a detoxification enzyme specialized in N-linked glucuronidation of many drugs and xenobiotics. It belongs to the UDP-glucuronosyltransferase (UGT) superfamily, which catalyzes the glucuronidation of molecules containing nucleophilic functional groups. UGT2B10 is of major importance in the conjugation and subsequent elimination of potentially toxic xenobiotics and endogenous compounds .

Its preferred substrates possess tertiary aliphatic amines and heterocyclic amines, such as tobacco carcinogens and several antidepressants and antipsychotics . UGT2B10 exhibits higher affinity than UGT1A4 for numerous substrates, making it potentially the more important enzyme for metabolism of these compounds in vivo . The study of UGT2B10 is particularly relevant due to clinically significant polymorphisms that can lead to large exposure differences for UGT2B10 substrates, potentially limiting their developability as marketed drugs .

What types of UGT2B10 antibodies are available for research?

Based on the available information, researchers have access to several types of UGT2B10 antibodies:

• Rabbit Polyclonal Antibodies: Such as ab225931, which is suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) and reacts with human samples. The immunogen corresponds to a recombinant fragment protein within Human UDP-glucuronosyltransferase 2B10 aa 1-300 .

• In-house Produced Antibodies: Such as the rabbit polyclonal anti-UGT2B10 #1845, produced against GST-UGT2B11 (aa 60-140), which has been used for immunodetection of UGT2B10 in HepG2 and HEK293 cell models .

• Commercial Monoclonal Antibodies: Such as the monoclonal anti-UGT2B10 antibody (ab57685; Abcam), which has been used for immunoprecipitation in human liver samples .

What are the validated applications for UGT2B10 antibodies?

UGT2B10 antibodies have been validated for several research applications:

It's important to note that antibody cross-reactivity has been observed. Analysis revealed that antibody #1845 detects UGT2B10, UGT2B11, and less efficiently UGT2B28 .

How can UGT2B10 antibodies be used to study alternative splicing variants?

Research has established that alternative splicing (AS) constitutes a means to regulate steady-state levels of UGT2B10 and enzyme activity. To study alternative transcript expression:

• First, establish the transcriptome of UGT2B10 in your tissue of interest. In normal liver tissue, 10 AS transcripts represented 50% of the UGT2B10 transcriptome .

• Use reverse-transcription PCR analysis of UGT2B10 transcripts to identify specific variants. Design primers to target regions of interest, particularly those involving novel exonic sequences .

• For protein-level detection of alternative variants:

  • Generate expression vectors from UGT2B10_v1 constructs using site-directed mutagenesis

  • Create tagged versions (e.g., V5-his tag) for immunoprecipitation studies

  • Express these constructs in UGT-negative (HEK293) or UGT-positive (HepG2) cell lines

  • Use Western blotting with specific antibodies to detect expression levels

• For quantitative analysis of alternative splicing, consider MS-coupled multiple reaction monitoring (MRM) approaches to detect peptides unique to alternative UGT2B10 variants .

What methodologies can be used to study UGT2B10 polymorphisms with antibody-based approaches?

UGT2B10 exhibits clinically relevant polymorphisms, including a null activity splice site mutation common in African populations. To study these polymorphisms:

• Identify individuals with relevant polymorphisms through genotyping.

• Utilize human liver microsomes (HLM) from phenotypically poor metabolizer donors as a model system. The activity difference between pooled HLM and HLM from a phenotypically UGT2B10 poor metabolizer donor can be exploited to identify UGT2B10-selective substrates .

• Perform comparative immunoblotting between normal and polymorphic samples to assess protein expression levels.

• Conduct immunoprecipitation followed by mass spectrometry to confirm the presence of specific UGT2B10 variants.

• Apply glucuronidation assays with specific substrates to functionally characterize the impact of polymorphisms on enzymatic activity.

This approach has been successful in identifying compounds like dothiepin, cidoxepin, cyclobenzaprine, azatadine, cyproheptadine, bifonazole, and asenapine as selective UGT2B10 substrates .

How can UGT2B10 antibodies be used in drug development research?

UGT2B10 antibodies are valuable tools in drug development for identifying compounds that might be significantly affected by UGT2B10 polymorphisms:

These approaches provide rapid and sensitive ways to evaluate whether a potential drug candidate cleared via glucuronidation will be sensitive to UGT2B10 polymorphisms in vivo .

What are the optimal conditions for immunohistochemistry using UGT2B10 antibodies?

For optimal immunohistochemistry results with UGT2B10 antibodies:

• Sample Preparation:

  • Use paraffin-embedded tissue sections

  • Perform appropriate antigen retrieval methods

  • Ensure tissues are properly fixed

• Antibody Selection and Dilution:

  • For rabbit polyclonal antibody ab225931, use a 1/100 dilution

  • This has been validated for human ovarian cancer and cervical cancer tissues

• Detection System:

  • Use an appropriate secondary antibody and detection system compatible with rabbit IgG

  • Include positive controls (such as human ovarian or cervical cancer tissues)

  • Include negative controls (primary antibody omitted)

• Interpretation:

  • Evaluate staining specificity by examining subcellular localization

  • UGT2B10 is typically localized to the endoplasmic reticulum

What are the best practices for immunoprecipitation of UGT2B10?

For effective immunoprecipitation of UGT2B10:

• Sample Preparation:

  • For human liver S9 fraction (8 mg of proteins), lyse for 45 minutes on ice in 4 ml of lysis buffer containing:

    • 0.05 M Tris-HCl (pH 7.4)

    • 0.15 M NaCl

    • 1% (w/v) Igepal CA-630

    • 1 mM dithiothreitol

    • Complete protease inhibitor cocktail

  • Centrifuge lysates for 15 minutes at 13,000 g

• Immunoprecipitation:

  • Use 10 μg of monoclonal anti-UGT2B10 (ab57685; Abcam)

  • Incubate for 1 hour at 4°C on an orbital shaker

  • Capture protein complexes by overnight incubation at 4°C with protein G–coated magnetic beads (200 μl of Dynabeads)

• Washing and Analysis:

  • Wash beads in lysis buffer and with 50 mM ammonium bicarbonate

  • For tryptic digestion and MS analysis, follow protocols as described in the literature

How can UGT2B10 protein half-life be determined using antibodies?

To determine UGT2B10 protein half-life:

• Treatment:

  • Treat cells expressing UGT2B10 with cycloheximide (20 μg/ml) for 0–16 hours to inhibit protein synthesis

• Sample Collection:

  • Wash cells in phosphate-buffered saline

  • Collect cells and lyse by scraping in lysis buffer

• Protein Analysis:

  • Perform immunoblotting using anti-UGT2B10 antibodies (such as #1845)

  • Quantify protein levels by densitometry at each time point

  • Plot protein levels versus time on a semi-logarithmic scale

  • Calculate half-life from the slope of the resulting line

• Validation:

  • Perform duplicate experiments to ensure reproducibility

  • Collect RNA samples to verify that protein degradation (rather than transcriptional changes) is being measured

How can cross-reactivity issues with UGT2B10 antibodies be addressed?

Analysis has revealed that some antibodies, such as #1845, detect not only UGT2B10 but also UGT2B11 and less efficiently UGT2B28 . To address cross-reactivity:

• Specificity Testing:

  • Test antibodies against recombinant UGT isoforms to identify cross-reactivity

  • Use control samples from UGT2B10-knockout systems or cells not expressing UGT2B10

• Optimization Strategies:

  • Adjust antibody concentration to minimize non-specific binding

  • Modify blocking conditions to reduce background

  • Consider pre-adsorption with recombinant proteins of cross-reactive UGTs

• Alternative Approaches:

  • Use mass spectrometry-based detection methods for definitive identification

  • Employ peptide-specific antibodies targeting unique regions of UGT2B10

  • Consider RNA-based detection methods (qPCR) in parallel for confirmation

What are the best methods to validate UGT2B10 antibody specificity?

To validate UGT2B10 antibody specificity:

• Cell and Tissue Models:

  • Compare antibody reactivity in UGT-negative cells (such as HEK293) versus those expressing UGT2B10

  • Test antibody in tissues known to express high (liver) versus low levels of UGT2B10

  • Use siRNA knockdown of UGT2B10 to confirm specificity

• Molecular Approaches:

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Use tryptic digests of UGT2B10 analyzed by MS-coupled multiple reaction monitoring (MRM)

  • Confirm peptide identity by coinjection of isotopically labeled synthetic peptides

• Comparative Analysis:

  • Test multiple antibodies against the same target

  • Compare reactivity patterns across different detection methods

How can UGT2B10 antibodies be used to distinguish between closely related UGT isoforms?

Distinguishing between closely related UGT isoforms is challenging but can be achieved through:

• Epitope Selection:

  • Use antibodies raised against unique regions of UGT2B10 that differ from other UGT isoforms

  • Target non-conserved regions, particularly in the C-terminal domain

• Detection Methods:

  • Employ MS-MRM approaches focusing on peptides unique to UGT2B10

  • Design targeted assays for detection of signature peptides in tryptic digests

• Functional Validation:

  • Combine antibody detection with functional assays using selective substrates

  • UGT2B10 exhibits higher affinity than UGT1A4 for numerous substrates

  • Use compounds like dothiepin, cidoxepin, cyclobenzaprine, azatadine, cyproheptadine, bifonazole, and asenapine, which are selective UGT2B10 substrates

• Comparative Expression Analysis:

  • Analyze expression patterns in tissues with known differential expression of UGT isoforms

  • Use liver tissue, which shows the highest expression of UGT2B10

How are UGT2B10 antibodies used in cancer research?

UGT2B10 antibodies have been applied in cancer research in several ways:

• Expression Analysis:

  • Immunohistochemical analysis of paraffin-embedded human ovarian and cervical cancer tissues has been performed using UGT2B10 antibody (ab225931) at 1/100 dilution

  • This helps in understanding the role of UGT2B10 in cancer metabolism and drug resistance

• Hepatocellular Carcinoma Studies:

  • The UGT2B10 transcriptome has been studied in 44 hepatocellular carcinomas, where 10 alternative splicing transcripts represented approximately 50% of the UGT2B10 transcriptome

  • This suggests potential roles in cancer development or response to therapy

• Drug Metabolism Research:

  • UGT2B10 is involved in the metabolism of several drugs used in cancer treatment

  • Antibodies help in assessing expression levels and potential impact on therapeutic outcomes

What are the methodological considerations for studying UGT2B10 in drug metabolism studies?

When studying UGT2B10 in drug metabolism:

• Enzyme Source Selection:

  • Human liver microsomes (HLM) are commonly used

  • Consider using both pooled HLM and HLM from phenotypically UGT2B10 poor metabolizer donors for comparative analysis

• Substrate Selection and Assay Conditions:

  • Select appropriate substrates known to be metabolized by UGT2B10

  • For enzymatic assays in intact cells (in situ assays), both HEK293 (UGT negative) and HepG2 (UGT positive) cell models can be used

  • Use appropriate substrate concentrations (e.g., amitriptyline at 7.5 and 150 μM; levomedetomidine at 7.5 and 75 μM)

• Detection Methods:

  • High-performance liquid chromatography–tandem mass spectrometry is typically used for glucuronide quantification

  • Normalize results to the expression of the UGT2B10 enzyme in each cell model, determined by densitometry scanning of band intensity on immunoblots

• Experimental Controls:

  • Include commercial supersomes expressing UGT1A and UGT2B isoenzymes as controls

  • Use microsomes of UGT2B11-expressing HEK293 prepared in-house for specificity comparison