POGLUT1 Antibody

What is POGLUT1 and why is it important in 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. It specifically targets extracellular EGF repeats of proteins such as CRB2, F7, F9, and NOTCH2 . POGLUT1 functions as a positive regulator of Notch signaling by mediating O-glucosylation of Notch, which is critical for proper muscle development .

Research on POGLUT1 is particularly important because mutations in the POGLUT1 gene have been identified as the cause of limb-girdle muscular dystrophy (LGMD R21; OMIM# 617232) . Additionally, POGLUT1 plays essential roles in early development, particularly in gastrulation, by mediating the O-glucosylation of CRB2, which is required for CRB2 localization to the cell membrane .

What applications are POGLUT1 antibodies suitable for?

Based on the available data, POGLUT1 antibodies are suitable for multiple research applications:

• Western blot analysis (WB): Commercial antibodies typically work at dilutions of 0.04-0.4 μg/ml or 0.25-0.5 μg/ml .

• Immunohistochemistry (IHC): Effective at dilutions of 1:200-1:500 .

• Immunohistochemistry on paraffin-embedded sections (IHC-P): Works at dilutions of 1:200-1:500 or 2-5 μg/ml .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Typically used at 5 μg/ml .

Different antibodies may have specific optimized conditions, so it's important to check the manufacturer's recommendations for the specific antibody being used.

How should I prepare samples for POGLUT1 antibody detection?

Sample preparation varies depending on the application:

For Western blot analysis:

• Dissect tissues in ice-cold PBS and snap freeze on dry ice.

• Lyse samples in RIPA buffer with protease inhibitors.

• Equilibrate lysates on ice for 20 minutes.

• Sonicate samples (3 × 30 seconds).

• Incubate lysates for 10 minutes on ice.

• Centrifuge to remove cell debris.

• Mix supernatant with 2× SDS loading dye (1:1) and load on SDS-PAGE .

For Immunohistochemistry:

• Fix tissues in 4% paraformaldehyde (PFA) overnight at 4°C for in situ hybridization or for one hour at room temperature for immunostaining.

• Embed tissues in OCT (optimal cutting temperature) compound.

• Cryosection at 10-12 μm thickness.

• Dilute primary antibodies in blocking buffer and incubate overnight at 4°C.

• Dilute secondary antibodies in blocking buffer and incubate for 1 hour at room temperature .

How can I use POGLUT1 antibodies to study Notch signaling defects?

POGLUT1 antibodies can be valuable tools for investigating Notch signaling defects through several approaches:

• Comparative Analysis of NICD Levels: Use antibodies against both POGLUT1 and the Notch1 intracellular domain (NICD) to assess correlation between POGLUT1 expression/activity and Notch signaling. Research has shown that POGLUT1 mutations lead to remarkable reduction in NICD levels .

• Co-detection Method:

  • Use anti-POGLUT1 antibodies to detect enzyme expression/localization

  • Use antibodies that specifically recognize the γ-secretase cleaved (S3 cleaved, Val1744) active NOTCH1 to assess pathway activation

  • Compare full-length unprocessed Notch (using antibodies to the intracellular domain) with the active form

• Quantitative Western Blot Protocol:

  • Prepare whole embryo/tissue lysates in RIPA buffer with protease inhibitors

  • Run standard SDS-PAGE and transfer to membrane

  • Probe with anti-cleaved-NOTCH1 (Val 1744) at 1:1000 dilution

  • Probe parallel samples with anti-NOTCH1 (detecting both full-length and cleaved forms) at 1:1000 dilution

  • Quantify the ratio of cleaved to total Notch to assess signaling efficiency

This approach has revealed that in POGLUT1-mutant models, active NOTCH1 is greatly reduced (6.9 ± 1.3 fold), while the full-length unprocessed form becomes more abundant .

What controls should I include when studying POGLUT1 mutations?

When studying POGLUT1 mutations, several controls are essential:

• Wild-type Controls: Always include wild-type POGLUT1 expression vectors when testing mutant forms to establish baseline activity and expression .

• Empty Vector Controls: Include empty vector transfections as negative controls to account for background signal and non-specific effects .

• Revertant Controls: Generate revertant constructs by reverting mutant sequences back to wild-type to confirm that observed defects are specifically due to the intended mutations rather than unintended mutations generated during PCR mutagenesis .

• Secretion Controls: When studying secreted forms of POGLUT1, include an independent secretion marker (such as IgG) whose secretion doesn't depend on POGLUT1-mediated O-glucosylation .

• Functional Validation: Test multiple functional outcomes of POGLUT1 activity, including:

  • Enzymatic activity assays

  • Protein stability measurements

  • Effects on downstream targets like Notch signaling components

  • Changes in α-dystroglycan glycosylation, which has been shown to be affected in POGLUT1 mutations

How can I assess the impact of POGLUT1 mutations on enzyme activity?

To assess the impact of POGLUT1 mutations on enzyme activity, researchers have established effective biochemical and cell-based assays:

• Site-directed Mutagenesis Protocol:

  • Generate POGLUT1 mutants using standard PCR-based site-directed mutagenesis

  • Use a pcDNA4-based wild-type POGLUT1-MycHis6 expression vector or a pTracer-based POGLUT1-FLAG expression vector as template

  • Design specific primers for each mutation of interest

  • Confirm successful incorporation of mutations by DNA Sanger sequencing

• Expression Analysis:

  • Transiently transfect HEK293T cells with wild-type and mutant POGLUT1 expression vectors using PEI

  • Include empty vector as negative control

  • Examine expression and secretion of POGLUT1 proteins in cell lysates and culture media by western blot analysis using anti-Myc antibody

• Functional Assays:

  • Co-transfect expression vectors encoding mouse Notch1 EGF1-18-MycHis6 and wild-type or mutant forms of POGLUT1-MycHis6 in wild-type or POGLUT1 knockout HEK293T cells

  • Include IgG expression vector as secretion control

  • Detect Notch1 EGF1-18-MycHis proteins, POGLUT1 proteins, and IgG in culture media and cell lysates by western blot

  • Assess changes in substrate modification through altered mobility on SDS-PAGE or through specialized glycosylation detection methods

What is the optimal protocol for immunohistochemistry using POGLUT1 antibodies?

Based on the search results, here is an optimized protocol for immunohistochemistry with POGLUT1 antibodies:

For Standard Tissue Sections:

• Dissect tissues in ice-cold PBS-BSA.

• Fix in 4% PFA for one hour at room temperature.

• Embed in OCT and cryosection at 10-12 μm thickness.

• Prepare blocking buffer (typically 10% goat serum, 5% BSA, 0.3% Triton-X100 in PBS).

• Dilute POGLUT1 primary antibody in blocking buffer at 1:200-1:500 dilution .

• Incubate sections with primary antibody overnight at 4°C.

• Wash sections thoroughly with PBS.

• Incubate with appropriate secondary antibody diluted in blocking buffer for 1 hour at room temperature.

• Include DAPI in the secondary incubation for nuclear counterstaining.

• Mount slides and image using confocal microscopy .

For Whole-Mount Specimens:

• Dissect specimens in ice-cold PBS-BSA.

• Fix overnight in 4% PFA/PBS at 4°C.

• Dehydrate in methanol and store at -20°C overnight.

• Rehydrate and perform antigen unmasking in appropriate unmasking solution at 98°C for 10 minutes.

• After cooling to room temperature, wash in MilliQ water.

• Incubate in acetone at -20°C for 8 minutes.

• Wash and incubate in blocking buffer overnight at 4°C.

• Incubate with primary antibody for 2 days.

• Following 4-5 washes with blocking buffer, incubate in secondary antibody overnight at 4°C.

• Wash extensively before mounting in glass-bottom dishes for confocal imaging .

How should I troubleshoot non-specific binding when using POGLUT1 antibodies?

When encountering non-specific binding with POGLUT1 antibodies, consider the following troubleshooting steps:

• Optimize Antibody Concentration:

  • Test a range of antibody dilutions. For Western blot, try 0.04-0.4 μg/ml; for IHC, try 1:200-1:500 .

  • Non-specific binding often improves at higher dilutions (lower antibody concentrations).

• Improve Blocking Conditions:

  • Use a more stringent blocking buffer (e.g., 10% goat serum, 5% BSA, 0.3% Triton-X100 in PBS) .

  • Extend blocking time to overnight at 4°C before primary antibody incubation.

• Include Appropriate Controls:

  • Use tissues or cells known to be negative for POGLUT1 expression.

  • Include a no-primary antibody control to assess secondary antibody specificity.

  • If available, use POGLUT1 knockout samples as negative controls.

• Modify Washing Procedure:

  • Increase the number and duration of washes after primary and secondary antibody incubations.

  • Use more stringent wash buffers (higher salt concentration or addition of mild detergents).

• Pre-adsorb Primary Antibody:

  • Incubate the diluted primary antibody with tissues or cells that lack the target but contain potentially cross-reacting proteins.

  • Remove any bound antibodies by centrifugation before using the supernatant for the actual experiment.

What are the best methods to quantify POGLUT1 expression levels?

For accurate quantification of POGLUT1 expression levels, the following methods are recommended:

• Western Blot Quantification:

  • Use standardized protein loading (verified by housekeeping protein detection).

  • Include a standard curve of recombinant POGLUT1 protein at known concentrations.

  • Capture images within the linear range of detection.

  • Use densitometry software to quantify band intensity.

  • Express results as relative to controls or as absolute quantities based on standard curves.

• Immunohistochemistry Quantification:

  • Use consistent staining protocols across all samples.

  • Capture images under identical exposure settings.

  • Analyze using software like ImageJ to measure:

    • Staining intensity (integrated density)

    • Percent positive cells

    • Subcellular localization patterns

• Flow Cytometry:

  • For cell suspensions, perform intracellular staining for POGLUT1.

  • Use appropriate isotype controls.

  • Quantify based on median fluorescence intensity or percent positive cells.

• Real-time PCR (complementary to protein detection):

  • Design specific primers for POGLUT1 mRNA.

  • Use appropriate reference genes for normalization.

  • Apply the 2^-ΔΔCt method for relative quantification.

• Mass Spectrometry-based Quantification:

  • For absolute quantification, use targeted proteomics approaches such as selected reaction monitoring (SRM) or parallel reaction monitoring (PRM).

  • Include isotopically labeled peptide standards for POGLUT1.

How can POGLUT1 antibodies be used to study muscular dystrophies?

POGLUT1 antibodies can be valuable tools in studying muscular dystrophies, particularly limb-girdle muscular dystrophy R21 (LGMD R21) which is caused by POGLUT1 mutations:

• Diagnostic Applications:

  • POGLUT1 antibodies can help diagnose LGMD R21 by detecting altered POGLUT1 expression or localization in muscle biopsies.

  • The combined approach of detecting POGLUT1, NICD, and α-dystroglycan glycosylation can provide a comprehensive diagnostic profile for LGMD R21 .

• Mechanistic Studies:

  • POGLUT1 antibodies allow for the investigation of downstream effects of POGLUT1 mutations.

  • Research has shown that POGLUT1 muscular dystrophy displays a characteristic "inside-to-outside" fatty degeneration pattern .

  • POGLUT1 antibodies can help correlate enzyme expression/function with this specific pattern.

• Satellite Cell Analysis Protocol:

  • Isolate satellite cells from muscle biopsies.

  • Use POGLUT1 antibodies in combination with PAX7 antibodies (a satellite cell marker).

  • Assess how POGLUT1 mutations affect the satellite cell pool, which has been reported to be reduced in POGLUT1 muscular dystrophy .

  • Study the impact on muscle cell proliferation and differentiation.

• Notch Signaling Assessment:

  • Use POGLUT1 antibodies together with antibodies against Notch signaling components.

  • Quantify NICD levels, which are remarkably decreased in muscle biopsies from patients with POGLUT1 mutations .

  • Establish the relationship between POGLUT1 function, Notch signaling impairment, and muscular dystrophy.

What are the key considerations when studying POGLUT1 in developmental contexts?

When studying POGLUT1 in developmental contexts, researchers should consider:

• Temporal Expression Analysis:

  • POGLUT1 plays critical roles in early development, particularly during gastrulation .

  • Use POGLUT1 antibodies to track expression patterns across developmental stages.

  • Compare with expression of Notch pathway components to establish functional relationships.

• Tissue-Specific Effects:

  • POGLUT1 functions in multiple tissues with potentially different roles.

  • In muscle development, POGLUT1 regulates Notch signaling .

  • In early embryonic development, POGLUT1 mediates O-glucosylation of CRB2, affecting its localization to the cell membrane .

• Experimental Model Selection:

  • Different models offer unique advantages for studying POGLUT1:

    • HEK293T cells for biochemical assays and protein production

    • Drosophila indirect flight muscle for in vivo functional studies

    • Mouse embryos for developmental analysis

    • Human patient-derived cells for disease-relevant mechanisms

• Combined Assessment Protocol:

  • For embryonic studies, use whole-mount immunostaining for active NOTCH1:

    • Fix embryos overnight in 4% PFA/PBS at 4°C

    • Perform antigen unmasking at 98°C

    • Incubate with anti-cleaved NOTCH1 antibody (1:1000)

    • Process for confocal imaging

  • Complement with in situ hybridization for Notch target genes (Hes5, Lunatic Fringe, Hes7)

  • Analyze POGLUT1 expression in the same developmental contexts

How can POGLUT1 antibodies be used in glycobiology research?

POGLUT1 antibodies offer unique opportunities for advancing glycobiology research:

• Enzyme-Substrate Interaction Studies:

  • Use POGLUT1 antibodies in co-immunoprecipitation assays to identify novel substrates.

  • Combine with mass spectrometry to characterize O-glucosylation sites.

  • Known substrates include proteins with the C-X-S-X-P-C consensus sequence in EGF repeats, such as Notch, CRB2, F7, and F9 .

• Glycosylation Pattern Analysis:

  • POGLUT1 antibodies can help correlate enzyme levels with specific glycosylation patterns.

  • This is particularly relevant for studying α-dystroglycan glycosylation, which is reduced in POGLUT1 muscular dystrophy .

• Structure-Function Relationships:

  • POGLUT1 has dual glycosyltransferase activity, transferring both glucose and xylose .

  • Antibodies recognizing different epitopes can help map functional domains.

  • This information is valuable for understanding how different mutations affect specific enzymatic functions.

• Glycosylation Pathway Crosstalk:

  • POGLUT1 antibodies can help investigate potential interactions between O-glucosylation and other glycosylation pathways.

  • This may reveal new insights into the regulation of complex glycosylation networks.

What novel approaches combine POGLUT1 antibodies with other technologies?

Innovative research approaches combining POGLUT1 antibodies with other technologies include:

• CRISPR-Based Functional Genomics:

  • Generate POGLUT1 knockout or knockin cell lines using CRISPR-Cas9.

  • Use POGLUT1 antibodies to verify gene editing efficiency.

  • Study the effects of specific mutations by introducing them into endogenous loci.

  • This approach has been used to create POGLUT1 knockout HEK293T cells for functional studies .

• Proximity Labeling Combined with Mass Spectrometry:

  • Fuse POGLUT1 to BioID or APEX2 enzymes.

  • Identify proximal proteins through biotinylation.

  • Verify interactions using POGLUT1 antibodies.

  • This can reveal transient or weak interactions in the native cellular context.

• Super-Resolution Microscopy:

  • Use fluorescently labeled POGLUT1 antibodies for super-resolution imaging.

  • Study subcellular localization with nanometer precision.

  • Investigate co-localization with substrates and other glycosylation machinery.

• Patient-Derived Organoids:

  • Develop organoids from patients with POGLUT1 mutations.

  • Use POGLUT1 antibodies to compare expression and localization with healthy controls.

  • Test potential therapeutic approaches targeting the Notch pathway.

How can I integrate POGLUT1 antibody data with other -omics approaches?

Integration of POGLUT1 antibody data with other -omics approaches can provide comprehensive insights:

• Multi-omics Integration Framework:

  • Combine POGLUT1 protein expression data (using antibodies) with:

    • Transcriptomics (RNA-seq) to correlate protein with mRNA levels

    • Glycoproteomics to identify and quantify O-glucosylated proteins

    • Metabolomics to assess changes in UDP-glucose/UDP-xylose metabolism

    • Phenomics to connect molecular changes with cellular/organismal phenotypes

• Network Analysis Protocol:

  • Use POGLUT1 antibodies to quantify protein expression across conditions.

  • Integrate with interaction data from co-immunoprecipitation experiments.

  • Apply network algorithms to identify functional modules affected by POGLUT1 dysregulation.

  • This approach can reveal non-obvious connections between POGLUT1 and other biological processes.

• Single-cell Analysis Approach:

  • Combine single-cell RNA-seq with immunofluorescence using POGLUT1 antibodies.

  • Map heterogeneity in POGLUT1 expression across cell populations.

  • Correlate with cell-specific Notch signaling activities.

  • This is particularly relevant for developmental studies and understanding tissue-specific effects.

• Longitudinal Studies in Disease Models:

  • Track POGLUT1 expression and localization over time using antibodies.

  • Correlate with disease progression markers.

  • Integrate with -omics data from the same timepoints to build predictive models.

  • This approach is valuable for understanding the temporal dynamics of POGLUT1-related pathologies.