pm20d1.1 Antibody

What is PM20D1 and what are its confirmed physiological functions?

PM20D1 (peptidase M20 domain containing 1) is a secreted enzyme that functions as a bidirectional NAA (N-fatty acyl amino acid) synthase/hydrolase, regulating endogenous N-fatty acyl amino acid tissue and circulating levels . The protein has an observed molecular weight of 55-60 kDa and is encoded by the gene with ID 148811 (NCBI) . PM20D1 plays a significant role in several physiological processes:

• Regulation of N-acyl amino acid metabolism

• Association with lipoprotein particles in circulation

• Involvement in metabolic regulation

• Potential role in nociception (pain perception)

Research has demonstrated that PM20D1 circulates in tight association with both low- and high-density lipoprotein particles, which serve as co-activators of PM20D1 activity in vitro and affect N-acyl amino acid biosynthesis in vivo .

What are the established species reactivity profiles for PM20D1 antibodies?

According to validated research, PM20D1 antibodies have confirmed reactivity with samples from the following species:

• Human

• Mouse

This reactivity has been validated across multiple experimental applications including Western blotting, Cytometric bead array, and Indirect ELISA . When designing experiments with PM20D1 antibodies, researchers should consider that while cross-reactivity may exist with other species, experimental validation would be necessary before application to samples from species not listed above.

What applications have been validated for PM20D1 antibodies?

PM20D1 antibodies have been successfully validated in several research applications:

Researchers should note that a PM20D1 antibody can be obtained in PBS-only storage buffer (BSA and azide free) at a concentration of 1 mg/mL, which makes it particularly suitable for conjugation and use in specialized applications such as multiplex assays requiring matched pairs .

How can PM20D1 antibodies be utilized to study Alzheimer's disease progression?

PM20D1 has been identified as a methylation QTL (mQTL) in Alzheimer's disease with dynamic methylation patterns that change throughout disease progression . Studies have shown that:

• Patients with Mild Cognitive Impairment (MCI) consistently display promoter hypomethylation at the PM20D1 locus

• This hypomethylation becomes more prominent in patients with mild to moderate AD

• As the disease progresses to advanced stages, the pattern reverses to hypermethylation in late-stage AD

Researchers can use PM20D1 antibodies in longitudinal studies to correlate protein expression levels with these methylation changes. This approach allows for tracking the relationship between epigenetic modifications and protein expression throughout disease progression, potentially identifying stage-specific biomarkers.

The following methodological approach is recommended:

• Collect peripheral blood samples at different disease stages

• Perform methylation analysis using bisulfite sequencing

• Correlate methylation patterns with PM20D1 protein levels using antibody-based detection

• Compare findings with neuropathological staging (such as Braak scores)

This comprehensive approach may help develop PM20D1 as a potential blood-based biomarker for AD progression .

What is the relationship between PM20D1 and lipoprotein particles, and how should this influence experimental design?

Research has revealed that PM20D1 circulates exclusively in association with lipoprotein particles in both mice and human plasma . This important finding has significant implications for experimental design:

• 91% of PM20D1 was found localized to APOB+ fractions

• The remaining 9% was detected on APOA1+ fractions

• PM20D1 is completely absent in fractions where the majority of plasma proteins elute

When designing experiments to detect or isolate PM20D1 from plasma or serum samples, researchers should:

• Consider lipoprotein fractionation methods (such as FPLC) to isolate PM20D1-containing fractions

• Include lipoprotein markers (APOB, APOA1) as controls in immunoblotting experiments

• Be aware that traditional protein isolation methods might not efficiently capture lipoprotein-associated PM20D1

• Consider the effects of lipoprotein levels on PM20D1 activity when comparing between patient groups

The tight association between PM20D1 and lipoproteins suggests that lipid metabolism may influence PM20D1 function, which should be taken into account when interpreting experimental results or designing interventional studies .

How does PM20D1 expression change in metabolic disorders such as gestational diabetes mellitus?

Studies have demonstrated that PM20D1 is significantly down-regulated in patients with gestational diabetes mellitus (GDM) compared to healthy pregnant individuals . This finding suggests PM20D1 may play a role in metabolic regulation during pregnancy.

Research findings showed:

• Markedly lower expression of PM20D1 in GDM patients compared to healthy controls

• Significant negative correlations between PM20D1 levels and inflammatory markers in the second trimester of pregnancy:

  • CRP (r = -0.507, p < 0.001)

  • IL-1β (r = -0.807, p < 0.001)

  • IL-6 (r = -0.791, p < 0.001)

  • TNF-α (r = -0.807, p < 0.001)

These correlations suggest that PM20D1 may be involved in inflammatory processes associated with metabolic disorders. Researchers studying PM20D1 in metabolic contexts should consider including measurements of inflammatory markers to better understand the role of PM20D1 in disease pathophysiology .

What challenges exist in detecting endogenous PM20D1, and what alternative approaches can researchers employ?

A significant challenge in PM20D1 research is the reported lack of antibodies with sufficient sensitivity and selectivity for reliable measurement of endogenous PM20D1 levels . Researchers have employed alternative approaches:

• Surrogate measurements:

  • Using epitope-tagged PM20D1 (e.g., PM20D1-flag) for overexpression studies

  • Measuring N-acyl amino acid enzyme activity as a functional readout of PM20D1 presence

• Activity-based detection:

  • Targeted liquid chromatography-mass spectrometry (LC-MS) to measure N-acyl amino acids as functional products of PM20D1 activity

  • Monitoring specific N-acyl amino acids like N-oleoyl-leucine and N-oleoyl-phenylalanine, which have been shown to increase 5.8-fold and 10.3-fold respectively with PM20D1 overexpression

• Protein fractionation approaches:

  • Using fast protein liquid chromatography (FPLC) to separate lipoprotein fractions

  • Combining FPLC with enzyme activity assays to localize PM20D1 activity

When designing experiments targeting endogenous PM20D1, researchers should consider these alternative approaches and potentially combine multiple methods to strengthen their findings.

What protocol optimizations are recommended for using PM20D1 antibodies in Western blotting?

Based on validated research protocols, the following optimizations are recommended for Western blotting with PM20D1 antibodies:

• Sample preparation:

  • For serum/plasma samples: Deplete serum albumin using methods such as Bio-Rad DEAE Affi-Gel Blue to reduce background

  • Extract proteins using RIPA buffer for optimal solubilization

  • Load approximately 30 μg of protein per lane for optimal detection

• Electrophoresis and transfer:

  • Use 10% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membranes for best signal retention

• Antibody incubation:

  • Primary antibody: Anti-PM20D1 antibody (commercial antibodies have been validated for this application)

  • Secondary antibody: HRP-conjugated secondary antibody appropriate for the host species of the primary antibody

  • Include appropriate controls:

    • Positive control: Recombinant PM20D1 or sample with confirmed PM20D1 expression

    • Negative control: Sample from PM20D1 knockout or knockdown model

• Expected results:

  • The PM20D1 protein should be visualized at 55-60 kDa

  • Use GAPDH or another housekeeping protein as a loading control for normalization

What are the recommended methods for immunoaffinity purification of PM20D1 and its interacting partners?

For researchers investigating PM20D1 protein interactions, the following immunoaffinity purification protocol has been validated:

• Expression system setup:

  • Transduce cells (e.g., primary adipocytes) with adenoviruses expressing C-terminally flag-tagged PM20D1 construct

  • Use LacZ as a control for non-specific binding

• Sample collection:

  • Harvest conditioned media when cells are robustly expressing and secreting PM20D1-flag

  • Centrifuge to remove cellular debris

• Immunoaffinity purification:

  • Use a mild anti-FLAG immunoaffinity purification protocol to preserve protein-protein interactions

  • Visualize purified proteins by silver staining

  • Expect to see a ~60kDa band corresponding to PM20D1-flag along with interacting proteins

• Protein identification:

  • For comprehensive identification, label samples with tandem mass tags for quantitation

  • Pool samples and analyze by shotgun proteomics

  • Apply filtering criteria: ≥2-fold enrichment in PM20D1 samples and ≥2 peptides per protein

This approach has successfully identified multiple PM20D1 interacting proteins, including lipoprotein components, which has led to important discoveries about PM20D1's physiological context .

How should researchers interpret contradictory findings about PM20D1 methylation in different tissues and disease stages?

Research has revealed opposite directional changes in PM20D1 methylation patterns between peripheral blood and brain tissue, as well as between early and late stages of Alzheimer's disease . When facing such contradictions, researchers should consider:

• Tissue-specific epigenetic regulation:

  • Peripheral blood shows hypomethylation of PM20D1 promoter in MCI and early AD

  • Brain tissues (hippocampus and frontal cortex) show hypermethylation in advanced AD

  • These differences likely reflect tissue-specific regulation mechanisms

• Disease progression dynamics:

  • Longitudinal data shows that initial promoter hypomethylation of PM20D1 during MCI and early stage AD

  • This pattern reverses to eventual promoter hypermethylation in late stage AD

  • This suggests dynamic regulation throughout disease progression rather than a fixed pattern

• Methodological approach for resolving contradictions:

  • Use longitudinal sampling when possible to capture temporal changes

  • Include multiple tissue types when feasible

  • Correlate methylation changes with neuropathological staging (e.g., Braak scores)

  • Integrate methylation data with gene expression analysis to understand functional relevance

These guidelines help researchers develop a more comprehensive understanding of PM20D1 regulation across different contexts and avoid misinterpretation of seemingly contradictory results .

What is the relationship between PM20D1 expression and inflammatory markers, and how should this inform research design?

Studies have identified significant negative correlations between PM20D1 levels and several inflammatory markers, particularly in the context of gestational diabetes mellitus . This relationship should inform research design in the following ways:

When designing studies involving PM20D1:

• Include inflammatory markers as covariates:

  • The strong negative correlations suggest inflammation may influence PM20D1 expression

  • Including these markers helps control for inflammatory status as a confounding factor

• Consider gestational timing:

  • Correlations were much stronger in the second trimester compared to the third trimester

  • This suggests temporal sensitivity in the relationship between PM20D1 and inflammation

• Integrate metabolic parameters:

  • Consider measuring lipid profiles (TG, HDL-C, LDL-C) alongside PM20D1 and inflammatory markers

  • GDM patients showed altered lipid profiles that may interact with PM20D1 function:

    • Elevated triglycerides (3.02 ±0.49 vs 2.28 ±0.30 mmol/l, p < 0.001)

    • Reduced HDL-C (2.03 ±0.14 vs 2.22 ±0.21 mmol/l, p < 0.001)

    • Elevated LDL-C (3.56 ±0.35 vs 3.23 ±0.31 mmol/l, p < 0.001)

This integrated approach acknowledges the complex relationship between PM20D1, inflammation, and metabolism, allowing for more nuanced interpretation of research findings.

What controls should be included when using PM20D1 antibodies in different experimental applications?

To ensure reliable and interpretable results when using PM20D1 antibodies, the following controls should be included:

For Western Blotting:

• Positive control: Recombinant PM20D1 protein or overexpression lysate

• Negative control: Lysate from PM20D1 knockout/knockdown samples

• Loading control: Housekeeping protein such as GAPDH, β-actin, or tubulin

• Molecular weight marker: To confirm the expected 55-60 kDa band size

For Immunoprecipitation:

• Input sample: Pre-IP lysate to verify protein presence

• Isotype control: IgG from the same species as the PM20D1 antibody

• Negative control IP: Using an antibody against an unrelated protein

• Beads-only control: To identify non-specific binding to beads

For ELISA:

• Standard curve: Using recombinant PM20D1 protein

• Blank wells: Buffer only (no sample or antibody)

• Negative control: Sample known to lack PM20D1

• Spike-in controls: Known amounts of recombinant protein added to samples

For Functional Assays:

• Activity standard: Purified recombinant PM20D1 at known concentration

• Enzyme inhibition control: Heat-inactivated samples or known inhibitors

• Substrate controls: Various N-acyl amino acids to demonstrate substrate specificity

Including these controls helps validate antibody specificity, verify technical procedure success, and support accurate data interpretation.

How can researchers overcome the challenges of PM20D1 detection when studying its role in Alzheimer's disease?

Studying PM20D1 in Alzheimer's disease presents specific challenges due to dynamic methylation patterns and varying expression levels. Researchers can implement the following strategies:

• Multi-modal approach to PM20D1 assessment:

  • Combine protein detection (antibody-based) with gene expression analysis (qPCR)

  • Include epigenetic assessment (methylation analysis) of the PM20D1 promoter region

  • This comprehensive approach provides context for interpreting changes in PM20D1 levels

• Genotype-stratified analysis:

  • PM20D1 is a methylation and expression QTL coupled to an AD-risk associated haplotype defined by rs708727

  • Stratify subjects by genotype to control for genetic influence on PM20D1 regulation

  • This approach helps differentiate disease-related changes from genotype-dependent effects

• Longitudinal sampling when possible:

  • Given the dynamic changes in PM20D1 methylation throughout disease progression

  • Initial hypomethylation during MCI phases followed by hypermethylation in advanced AD

  • Single time-point measurements may be misleading without disease stage context

• Cross-tissue validation:

  • When feasible, compare findings between peripheral blood and brain tissue

  • Be aware that methylation patterns may differ between tissues

  • The relationship between peripheral and central PM20D1 regulation helps establish the utility of blood-based measurements as a potential biomarker

This multi-faceted approach addresses the complexity of PM20D1 regulation in Alzheimer's disease and increases the reliability of research findings.