POGLUT1 Antibody, HRP conjugated

What is POGLUT1 and why is it significant for research?

POGLUT1 is a dual specificity glycosyltransferase that catalyzes the transfer of glucose and xylose from UDP-glucose and UDP-xylose to serine residues found in the consensus sequence C-X-S-X-P-C within EGF repeats of various proteins, including Notch receptors . Its significance stems from:

• Critical role in Notch signaling pathway regulation

• Association with limb-girdle muscular dystrophy (LGMDR21)

• Function in muscle stem cell formation, maintenance, and homing

• Involvement in early embryonic development through CRB2 O-glucosylation

POGLUT1 specifically targets extracellular EGF repeats of proteins such as CRB2, F7, F9, and NOTCH2, making it a key research target for developmental biology and muscle disease studies .

What are the typical applications for POGLUT1 antibodies in research?

POGLUT1 antibodies are versatile tools employed in multiple experimental contexts:

These applications enable researchers to investigate POGLUT1's role in normal physiology and disease states, particularly in muscle development and Notch signaling contexts .

What is the optimal protocol for Western blot detection of POGLUT1?

Based on published protocols, the following methodology yields consistent POGLUT1 detection:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Determine protein concentration (Bradford or BCA assay)

  • Load 20-30 μg total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE gel

  • Transfer to PVDF membrane (nitrocellulose alternative)

• Immunodetection:

  • Block membrane with 5% non-fat milk in TBST (1 hour, RT)

  • Incubate with primary POGLUT1 antibody (1:100-1:2000 dilution, overnight at 4°C)

  • Wash 3× with TBST (10 minutes each)

  • Incubate with HRP-conjugated secondary antibody (1:5000, 1 hour, RT)

  • Wash 3× with TBST (10 minutes each)

  • Develop with enhanced chemiluminescence substrate

• Expected result:

  • POGLUT1 appears as a specific band at approximately 46 kDa

  • Expression is detectable in multiple cell types including HepG2, Jurkat, and IMR-32 cell lines

For optimal signal-to-noise ratio, titration of primary and secondary antibodies is recommended, with validation in appropriate positive control samples .

How should POGLUT1 enzymatic activity be assessed using antibody-based methods?

POGLUT1 enzymatic activity assessment combines antibody detection with functional assays:

• Protein expression and purification:

  • Express wild-type or mutant POGLUT1 with epitope tags (MycHis6, FLAG) in HEK293T cells

  • Purify from culture media using affinity chromatography

  • Confirm expression by Western blot with anti-tag or anti-POGLUT1 antibodies

• Enzymatic activity measurement:

  • Incubate purified POGLUT1 (100-200 ng) with:

    • 10 μM UDP-glucose donor substrate

    • 10 μM acceptor substrate (EGF repeat from human factor IX or Notch EGF repeats)

    • Reaction buffer (50 mM HEPES, pH 6.8)

    • 37°C for 1 hour

  • Quantify UDP release using UDP-Glo™ Glycosyltransferase Assay Kit

  • Analyze reaction products by reverse-phase HPLC and mass spectrometry

• Data interpretation:

  • Compare wild-type and mutant POGLUT1 activity levels

  • Assess activity across multiple substrate concentrations

  • Evaluate O-glucosylation of different EGF repeats (e.g., from Notch1, factor IX)

This approach has successfully characterized multiple POGLUT1 mutations, including D233E and R183W, revealing reduced but residual enzymatic activity in disease-associated variants .

How can POGLUT1 antibodies be utilized to investigate Notch signaling disruption in muscular dystrophy?

POGLUT1 antibodies enable multifaceted investigation of Notch pathway dysregulation in muscular dystrophy:

• Dual protein detection strategy:

  • Simultaneously analyze POGLUT1 and cleaved NOTCH1 (using val1744 antibody) from patient samples

  • Quantify relative expression levels by Western blot densitometry

  • Compare with healthy control tissues to establish correlation between POGLUT1 dysfunction and Notch signaling impairment

• Functional assessment in cell models:

  • Co-transfect expression vectors for Notch1 EGF1-18-MycHis6 with wild-type or mutant POGLUT1

  • Include IgG expression vector as secretion control

  • Detect proteins in culture media and cell lysates using anti-Myc and anti-human IgG antibodies

  • Evaluate O-glucosylation status and processing efficiency

• Tissue-specific expression analysis:

  • Perform immunohistochemistry on muscle biopsies from LGMDR21 patients

  • Assess POGLUT1 localization relative to markers of muscle stem cells (PAX7+)

  • Evaluate downstream Notch targets to measure pathway activity

This comprehensive approach has revealed that POGLUT1 mutations in LGMDR21 lead to impaired Notch signaling, reduced muscle stem cell abundance, and accelerated differentiation, which are probable mechanisms in disease pathophysiology .

What methodological approaches can distinguish between different POGLUT1 mutations using antibodies?

Distinguishing POGLUT1 mutations requires integrated antibody-based techniques:

• Expression analysis of mutant proteins:

  • Generate mutant POGLUT1 constructs (Y57C, R183W, I129T, R98W, C102F, W308L, D233E) using site-directed mutagenesis

  • Express in HEK293T cells and analyze by Western blot

  • Compare expression levels and secretion patterns to identify mutation-specific differences

• Revertant mutation analysis:

  • Create revertant constructs to confirm phenotype specificity

  • Express in HEK293T cells and compare with original mutants

  • Use Western blot with anti-POGLUT1 or anti-tag antibodies to assess expression

• Functional distinction strategy:

  • Measure enzymatic activity of purified mutant proteins

  • Test activity with multiple substrates (different EGF repeats)

  • Conduct kinetic analyses to determine specific effects on:

    • Substrate binding affinity

    • Catalytic efficiency

    • Protein stability

Research has shown that different mutations have distinct effects on POGLUT1 function. For example, the D233E mutation retains residual enzymatic activity while showing significantly lower efficiency across multiple substrates, whereas other mutations may affect protein folding or secretion .

What are optimal conditions for preventing non-specific binding when using HRP-conjugated secondary antibodies with POGLUT1?

Minimizing background requires optimization of several parameters:

• Blocking optimization:

  • Test different blocking agents:

    • 5% non-fat milk in TBST (standard)

    • 3-5% BSA in TBST (alternative for phospho-specific detection)

    • Commercial blocking buffers

  • Extend blocking time to 2 hours at room temperature for challenging samples

• Antibody dilution optimization:

  • Primary antibody: Test dilution range from 1:100 to 1:2000

  • HRP-conjugated secondary: Use 1:5000 for standard applications, adjust to 1:2000-1:10,000 based on signal strength

  • Consider antibody diluent composition (addition of 0.1-0.5% Tween-20 can reduce non-specific binding)

• Stringent washing protocol:

  • Increase wash steps (5× with TBST, 10 minutes each)

  • Use higher Tween-20 concentration in wash buffer (0.1-0.2%)

  • Consider adding low salt concentration (50-100mM NaCl) to wash buffer

• Development optimization:

  • Adjust exposure time to minimize background

  • Consider alternative substrates:

    • SuperSignal West Femto for high sensitivity

    • Standard ECL for moderate sensitivity

    • Chromogenic substrates for lower sensitivity but cleaner background

These approaches have been successfully applied in POGLUT1 research to achieve specific detection in various cell lines and tissue samples .

How can researchers validate POGLUT1 antibody specificity for advanced experimental applications?

Comprehensive validation approach includes:

• Genetic validation:

  • Use POGLUT1 knockout cell lines (POGLUT1 KO HEK293T) as negative controls

  • Compare signal between wild-type and knockout samples

  • Rescue experiments with POGLUT1 re-expression

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Compare signal between blocked and unblocked antibody

  • Gradually titrate blocking peptide to assess specificity threshold

• Multiple antibody validation:

  • Test different antibodies targeting distinct POGLUT1 epitopes:

    • N-terminal regions (e.g., Arg24-Leu388 region)

    • C-terminal regions (e.g., aa 300 to C-terminus)

    • Internal epitopes

  • Compare detection patterns across antibodies

• Cross-species reactivity assessment:

  • Test antibody in tissues/cells from different species

  • Compare with predicted conservation of epitope sequence

  • Evaluate background in non-target species as specificity indicator

These validation approaches ensure reliable results in advanced applications such as studying POGLUT1's role in developmental processes, muscle regeneration, and disease mechanisms .

How should researchers quantitatively analyze POGLUT1 expression in disease models using antibody-based detection?

Quantitative analysis of POGLUT1 requires systematic methodology:

• Standardized densitometry protocol:

  • Capture images using linear detection range of imaging system

  • Use consistent exposure times across experimental groups

  • Normalize POGLUT1 signal to loading control (GAPDH recommended)

  • Calculate relative expression using software like ImageJ or Image Studio Lite

• Statistical analysis framework:

  • Perform minimum of three biological replicates

  • Apply appropriate statistical tests:

    • t-test for two-group comparisons

    • ANOVA with post-hoc tests for multiple groups

    • Non-parametric alternatives for non-normal distributions

  • Consider p<0.05 significant with appropriate corrections for multiple testing

• Correlation with functional parameters:

  • Relate POGLUT1 levels to enzymatic activity measurements

  • Correlate with Notch pathway activity markers

  • Associate with phenotypic outcomes in disease models

• Multi-dimensional data integration:

  • Combine protein expression data with:

    • RNA expression analysis

    • Enzymatic activity measurements

    • Clinical/phenotypic outcomes

  • Apply multivariate analysis to identify key relationships

This approach has been successfully implemented in LGMDR21 research, revealing correlation between reduced POGLUT1 expression/activity and disease severity .

What methodological approaches can resolve contradictory POGLUT1 antibody results in different experimental systems?

Resolving contradictory results requires systematic troubleshooting:

• Sample preparation comparison:

  • Test multiple lysis buffers (RIPA, NP-40, Triton X-100)

  • Compare protein extraction efficiency from different tissues/cells

  • Evaluate impact of protease inhibitors on detection

• Epitope accessibility assessment:

  • Test native vs. denatured conditions

  • Evaluate impact of reducing agents on detection

  • Consider epitope masking by protein interactions or post-translational modifications

• Antibody cross-reactivity analysis:

  • Perform immunoprecipitation followed by mass spectrometry

  • Identify potential cross-reactive proteins

  • Use alternative antibodies targeting different epitopes

• System-specific optimization:

  • Cell-type specific protocol adjustments:

    • HepG2, Jurkat, and IMR-32 cells show consistent detection at ~46 kDa

    • Muscle tissue may require specialized extraction methods

  • Tissue-specific considerations:

    • Paraffin embedded sections require heat-induced epitope retrieval

    • Fresh tissue requires gentle fixation methods

By implementing this systematic approach, researchers have successfully resolved discrepancies in POGLUT1 detection across experimental systems, particularly in complex tissues like muscle where protein extraction can be challenging .