MGEA5 Antibody

What is MGEA5 and why is it important in cellular research?

MGEA5 (Meningioma-expressed antigen 5), also known as OGA, MEA5, or NCOAT, is a key enzyme in O-GlcNAc signaling pathways. It functions primarily as a glycosidase that removes O-linked N-acetylglucosamine (O-GlcNAc) modifications from serine and threonine residues of proteins. The dynamic modification of cytoplasmic and nuclear proteins by O-GlcNAc is catalyzed by the opposing actions of OGT (O-GlcNAc transferase), which adds the modification, and MGEA5, which removes it .

MGEA5 has significant research importance because:

• It regulates post-translational protein modifications affecting diverse cellular processes

• It has been identified as having both O-GlcNAcase domain and a putative histone acetyltransferase domain

• It deglycosylates a large and diverse number of proteins, including CRYAB, ELK1, GSDMD, LMNB1, and TAB1

• Its dysregulation has been implicated in various pathological conditions

How do I choose the appropriate MGEA5 antibody for my specific experimental needs?

Selecting the optimal MGEA5 antibody requires consideration of multiple factors:

Antibody format consideration matrix:

Key selection criteria:

• Application compatibility: Verify the antibody has been validated for your specific application. For example, antibody #14711-1-AP has been validated for multiple applications including WB, IHC, ICC, IF, IP, CoIP, ChIP, and RIP .

• Species reactivity: Ensure the antibody recognizes MGEA5 in your experimental species. Some antibodies (like A03465-1) react with human, mouse, and rat MGEA5 , while others (84722-3-PBS) may be human-specific .

• Isoform recognition: MGEA5 has multiple isoforms. Confirm which isoform(s) the antibody recognizes. Some detect only the full-length protein while others may detect multiple variants .

• Validated performance: Look for knockout validation data. Antibodies like those from Abcam (ab124807) and Bethyl have been validated in knockout models .

• Recombinant vs traditional: Consider recombinant antibodies (like ab124807 or 84722-4-RR) for improved lot-to-lot consistency .

What are the optimal dilution ratios for different applications of MGEA5 antibodies?

Optimal dilution ratios vary significantly based on the specific antibody and application. Below is a compilation of recommended dilutions from validated MGEA5 antibodies:

MGEA5 Antibody 14711-1-AP (Proteintech) :

• Western Blot: 1:2000-1:16000

• Immunoprecipitation: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunohistochemistry: 1:50-1:500

• Immunofluorescence/ICC: 1:50-1:500

• Flow Cytometry: 0.40 μg per 10^6 cells in a 100 μl suspension

MGEA5 Antibody 84722-4-RR (Proteintech) :

• Western Blot: 1:5000-1:50000

• Immunohistochemistry: 1:125-1:500

• Immunofluorescence/ICC: 1:200-1:800

MGEA5 Antibody (Cepham Life Sciences) :

• Western Blot: 1:200-1:1000

• Immunohistochemistry: 1:25-1:100

Methodological considerations:

• Always perform a dilution series to determine optimal conditions for your specific sample and experimental design

• For Western blot applications, consider using BSA instead of milk for blocking when using phospho-specific antibodies

• For IHC applications, test both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval to determine optimal conditions

• For quantitative applications, validate the linear dynamic range of the antibody signal

How can I troubleshoot non-specific binding when using MGEA5 antibodies in Western blotting?

Non-specific binding is a common challenge with MGEA5 antibodies, particularly given the discrepancy between calculated (102-103 kDa) and observed (130-140 kDa) molecular weights .

Step-by-step troubleshooting approach:

• Verify antibody specificity:

  • Use knockout or knockdown controls where available

  • Compare with a second validated antibody targeting a different epitope

  • Note that MGEA5 has been observed at both 75 kDa and 130 kDa in different cellular compartments

• Optimize blocking conditions:

  • Test different blocking agents (5% milk, 3-5% BSA)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to reduce background

• Adjust antibody concentration:

  • Use a more dilute antibody solution based on the recommended ranges

  • For 14711-1-AP, a 1:5000-1:8000 dilution often provides optimal signal-to-noise ratio

• Optimize washing steps:

  • Increase washing time or number of washes

  • Use TBS-T with 0.1% Tween-20 for washing

• Address post-translational modifications:

  • MGEA5 undergoes extensive post-translational modifications that affect observed molecular weight

  • Test samples with and without phosphatase treatment to assess contribution of phosphorylation

How can MGEA5 antibodies be effectively used to study subcellular localization of different isoforms?

MGEA5 exists in multiple isoforms with distinct subcellular localizations - a phenomenon critical to understanding its varied functions. Historical research has identified at least two forms: a 130 kDa cytoplasmic/cytoskeletal protein and a 75 kDa nuclear protein .

Methodological approach:

• Multiple detection methods combination:

  • Use immunofluorescence with confocal microscopy as primary approach

  • Validate findings with subcellular fractionation followed by Western blotting

  • Consider super-resolution microscopy for detailed localization studies

• Isoform-specific detection strategy:

  • Select antibodies recognizing specific domains (N-terminal vs. C-terminal)

  • The 14711-1-AP antibody can detect full-length MGEA5 while others may be more specific to particular variants

  • For the splice variant missing the putative acetyltransferase domain, select antibodies raised against the N-terminal region

• Co-localization analysis protocol:

  • Use nuclear markers (DAPI, lamin B1) and cytoskeletal markers (tubulin, actin)

  • Apply quantitative co-localization analysis (Pearson's coefficient, Manders' coefficient)

  • Consider live-cell imaging with tagged MGEA5 constructs to complement antibody studies

• Validation approaches:

  • Use cell fractionation (nuclear vs. cytoplasmic) followed by Western blotting

  • Compare results across multiple validated antibodies

  • Include knockout/knockdown controls

What approaches can be used to investigate the interactions between MGEA5 and its substrate proteins?

Investigating MGEA5-substrate interactions requires multiple complementary approaches to establish both physical interaction and functional relationship.

Recommended methodological workflow:

• Co-immunoprecipitation (Co-IP) strategy:

  • Use MGEA5 antibodies suitable for IP, such as 14711-1-AP or ab124807

  • Perform reciprocal Co-IP (pull down with substrate antibody, probe for MGEA5)

  • Consider performing under different cellular conditions (stress, inhibitor treatments)

  • Use appropriate controls (IgG control, lysate from MGEA5 knockout cells)

• Proximity ligation assay (PLA) implementation:

  • Allows visualization of protein interactions in situ

  • Use MGEA5 antibody from one species and substrate antibody from another species

  • Optimize fixation conditions to preserve transient interactions

• Functional O-GlcNAcylation assessment:

  • Use RL2 or CTD110.6 antibodies to detect global O-GlcNAcylation levels

  • Compare O-GlcNAcylation of specific substrates with and without MGEA5 modulation

  • Utilize MGEA5 inhibitors as complementary approach to antibody studies

• Mass spectrometry-based approaches:

  • Immunoprecipitate MGEA5 and identify binding partners

  • Use SILAC or TMT labeling to quantify changes in the MGEA5 interactome

  • Enrich for O-GlcNAcylated proteins and determine MGEA5-dependent changes

How can MGEA5 antibodies be utilized to study the enzyme's role in disease models?

MGEA5/OGA plays critical roles in various diseases through its regulation of O-GlcNAc cycling. Antibody-based approaches can provide valuable insights into disease mechanisms.

Disease model investigation approaches:

• Neurodegenerative disease models:

  • Analyze MGEA5 expression and localization in Alzheimer's and Parkinson's models

  • Use immunohistochemistry with MGEA5 antibodies (dilution 1:50-1:100) on brain tissue sections

  • Evaluate co-localization with disease-specific markers (tau, α-synuclein)

  • Compare MGEA5 levels in affected vs. unaffected brain regions

• Cancer models:

  • MGEA5 was originally identified in meningioma patients

  • Evaluate MGEA5 expression in tumor samples using immunohistochemistry (1:125-1:500 dilution)

  • Use tissue microarrays to compare expression across tumor types and stages

  • Correlate MGEA5 levels with patient outcomes and treatment response

• Metabolic disease models:

  • Analyze MGEA5 expression in insulin-responsive tissues

  • Compare expression and activity in normal vs. diabetic models

  • Use Western blotting to quantify MGEA5 levels in tissue samples

• Validation approaches:

  • Include multiple antibodies targeting different epitopes

  • Use genetic models (knockdown, knockout, overexpression)

  • Combine with functional assays (enzymatic activity measurements)

What methodological considerations are important when using MGEA5 antibodies in therapeutic antibody development research?

While this question enters territory that could include commercial applications, focusing on the methodological research aspects remains relevant for academic investigators developing potential therapeutic approaches.

Critical methodological considerations:

• Epitope specificity determination:

  • Map the exact epitope recognized by each antibody

  • For antibodies like 14711-1-AP, the immunogen was a MGEA5 fusion protein (Ag6405)

  • For E9C5U antibody, the immunogen corresponds to residues surrounding Lys545 of human MGEA5/OGA protein

  • Epitope specificity affects ability to inhibit enzymatic function

• Functional inhibition assessment:

  • Test antibodies for ability to inhibit MGEA5 enzymatic activity in vitro

  • Compare with established small-molecule inhibitors

  • Assess cell permeability of antibodies for intracellular targeting

• Cross-reactivity analysis:

  • Evaluate potential cross-reactivity with related glycosidases

  • Test against MGEA5 from multiple species if developing for animal models

  • Use knockout validation to confirm specificity

• Stability and modification analysis:

  • Evaluate antibody stability under various conditions

  • Consider engineering approaches to enhance therapeutic properties

  • Test functional properties of various antibody fragments

What approaches should be used to validate MGEA5 antibody specificity in different experimental systems?

Rigorous validation is essential for confident interpretation of MGEA5 antibody-based experiments. A comprehensive validation strategy incorporates multiple complementary approaches.

Multi-level validation strategy:

• Genetic validation approaches:

  • Knockout/knockdown validation: Compare signal in wild-type vs. MGEA5-deficient samples

  • Both ab124807 and Bethyl A304-345A antibodies have been validated using knockout models

  • Overexpression validation: Compare signal in control vs. MGEA5-overexpressing samples

  • siRNA titration: Demonstrate proportional reduction in signal with increasing siRNA

• Epitope-based validation:

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide

  • For antibodies like A03465-1, blocking peptide can be purchased and used for validation

  • Multiple antibody verification: Compare signals from antibodies targeting different epitopes

• Technical validation parameters:

  • Dilution linearity: Demonstrate proportional signal reduction with antibody dilution

  • Sample loading linearity: Show proportional signal increase with increasing sample loading

  • Batch-to-batch consistency: Compare results across different antibody lots

• Application-specific validation:

  • For IHC: Include positive and negative control tissues

  • For IF: Include subcellular localization controls based on known MGEA5 distribution

  • For IP: Verify enrichment by comparing input, flow-through, and elution fractions

How do post-translational modifications of MGEA5 affect antibody recognition, and how can these effects be evaluated?

MGEA5 itself undergoes post-translational modifications that can affect antibody recognition and experimental outcomes. Understanding and controlling for these effects is crucial for accurate interpretation.

Systematic evaluation approach:

• Identification of key modifications:

  • MGEA5 undergoes phosphorylation, which may explain the discrepancy between calculated (103 kDa) and observed (130-140 kDa) molecular weights

  • Other potential modifications include acetylation and ubiquitination

  • Determine if the antibody epitope contains modification sites

• Modification-dependent recognition assessment:

  • Treatment with phosphatases to remove phosphorylation

  • Compare antibody recognition before and after treatment

  • Use modification-specific antibodies in parallel to correlate modifications with recognition

• Context-dependent modification analysis:

  • Compare MGEA5 detection across different cell types and tissues

  • Evaluate effects of cellular stress conditions on antibody recognition

  • Consider cell cycle-dependent variations in modifications

• Technical approaches to control modification effects:

  • Use denaturing conditions in Western blotting to minimize conformation effects

  • Consider native vs. reduced/alkylated conditions in immunoprecipitation

  • For mass spectrometry validation, enrich for specific post-translational modifications