ME2 Antibody

What is ME2 and why is it an important research target?

ME2 (malic enzyme 2, NAD(+)-dependent, mitochondrial) is a genome-coded mitochondrial enzyme that catalyzes the conversion of malate to pyruvate while reducing NAD(+) to NADH. Its significance extends beyond basic energy metabolism to immune regulation and cancer biology. ME2 is crucial for NADPH production, lipogenesis, glutamine metabolism, and is involved in neuronal synthesis of the neurotransmitter γ-aminobutyric acid (GABA) . Recent research has revealed that ME2 affects fumarate levels, which regulate CD8+ T cell functionality and enhance antitumor immunity, making it a key focus in cancer metabolism research .

Additionally, ME2 has been linked to epilepsy syndromes through case-control and family-based association methods . The protein exists in two isoforms produced by alternative splicing with molecular weights of 65 kDa and 54 kDa, often observed simultaneously in experimental detection .

How do I select the appropriate ME2 antibody for my specific application?

Selecting the right ME2 antibody requires careful consideration of several factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IP, ELISA, IF)

• Species reactivity: Ensure the antibody recognizes ME2 in your model organism (human, mouse, rat)

• Isoform detection: Determine whether you need to detect specific or both ME2 isoforms

• Clonality: Consider whether a monoclonal or polyclonal antibody better suits your research needs

For example, if working with human tissue samples for immunohistochemistry, you might consider antibodies like 24944-1-AP which has been validated for human pancreatic cancer tissue and colon tissue with specific antigen retrieval recommendations . For studies requiring detection of both ME2 isoforms, antibodies such as those from Abcepta have been specifically validated to detect both the 64 kDa and 60 kDa forms .

When published data is available, prioritize antibodies with citation records in applications similar to yours, as this provides evidence of reliability in your specific context .

What are the optimal sample preparation protocols for ME2 detection in different applications?

Sample preparation requirements vary by application and tissue type:

For Western Blot (WB):

• Complete cell lysis is crucial as ME2 is localized to mitochondria

• Use mitochondrial isolation protocols when specifically studying mitochondrial fractions

• For whole cell lysates, RIPA buffer with protease inhibitors is generally effective

• Validated in multiple cell lines including HL-60, HeLa, Jurkat, K-562, LNCaP, HepG2, and NIH/3T3 cells

For Immunohistochemistry (IHC):

• Antigen retrieval is critical; most ME2 antibodies perform optimally with TE buffer pH 9.0

• Alternative protocol uses citrate buffer pH 6.0

• Paraffin-embedded human tissues (especially pancreatic cancer, colon, and liver cancer) have been successfully used

For Immunoprecipitation (IP):

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• HeLa cells have been specifically validated for IP applications

For Immunofluorescence (IF):

• Initial concentration of 20 μg/mL is recommended, followed by optimization

• Fixation with 4% paraformaldehyde preserves ME2 antigenicity

Confirming antibody specificity is crucial for reliable results. Implement these validation approaches:

• Peptide competition assay: Pre-incubate the antibody with excess ME2 peptide antigen to confirm signal reduction

• Knockout/knockdown controls: Use ME2 knockout cell lines or siRNA-treated cells as negative controls

• Cross-reactivity assessment: Test for signals in samples known to lack ME2 or use recombinant ME1 to test cross-reactivity

• Multiple antibody approach: Use multiple antibodies targeting different ME2 epitopes to confirm consistent patterns

• Observed vs. predicted MW comparison: Verify that observed molecular weights match the expected 65 kDa and 54 kDa bands for the two ME2 isoforms

Rigorously validating specificity is particularly important given the finding from peptide arrays and Internally Calibrated ChIP (ICeChIP) studies showing that antibody specificity can vary widely and often diverges across methods, with apparent specificity in peptide arrays and ICeChIP being only weakly correlated .

Why might I see multiple bands when using ME2 antibodies in Western blot?

Multiple bands in ME2 Western blots can occur for several legitimate reasons:

• Isoform detection: ME2 has two known isoforms (65 kDa and 54 kDa) produced by alternative splicing. Both are frequently detected simultaneously

• Post-translational modifications: ME2 can undergo modifications affecting migration patterns

• Degradation products: Improper sample handling may result in protein degradation

• Cross-reactivity: Some antibodies may detect related malic enzyme family members

To determine which explanation applies:

• Compare your observed band pattern with the reported molecular weights (54 kDa, 65 kDa)

• Verify using knockout/knockdown controls

• Use antibodies specifically validated to distinguish between isoforms or detect both

• Optimize sample preparation to minimize degradation

When in doubt, consult the antibody datasheet, as many manufacturers specifically note the expected pattern: "This protein has 2 isoforms produced by alternative splicing with the MW of 65 kDa and 54 kDa" .

What are the optimal storage conditions for maintaining ME2 antibody activity?

Proper storage is essential for maintaining antibody performance. Based on manufacturer recommendations across multiple antibody products:

• Store at -20°C for long-term stability (up to one year)

• For antibodies in solution with 50% glycerol, aliquoting is generally unnecessary for -20°C storage

• Short-term storage (up to three months) at 4°C is acceptable for some formulations

• Avoid repeated freeze-thaw cycles by preparing appropriate aliquots

• Some antibodies have specific formulations: "Store at 4°C in the dark. Do not freeze"

Always check product-specific recommendations, as storage buffers vary: "PBS with 0.02% sodium azide and 50% glycerol pH 7.3" is common , but formulations without preservatives may have different requirements .

How can ME2 antibodies be used to study cancer metabolism and potential therapeutic applications?

ME2 has emerged as a significant player in cancer metabolism, with implications for therapeutic development. Advanced research applications include:

• Metabolic profiling: Use ME2 antibodies in combination with metabolic flux analysis to understand how ME2 activity affects the NAD+/NADH ratio and ATP synthesis in cancer cells

• Drug discovery platforms: Employ ME2 antibodies to screen for and validate novel ME2 inhibitors, building on research showing that compounds like 5,5'-Methylenedisalicylic acid (MDSA) and embonic acid (EA) bind allosterically to ME2's fumarate-binding site

• Mechanism studies: Investigate how "ME2 overexpression increases pyruvate and NADH production while decreasing the cell's NAD+/NADH ratio," while inhibition has opposite effects

• Biomarker development: Evaluate ME2 expression in patient samples to determine correlations with disease progression or treatment response

Research has demonstrated that "ME2 silence or inhibiting ME2 activity with MDSA or EA decreases cellular respiration and ATP synthesis," suggesting therapeutic potential in targeting cancer cell metabolism .

What approaches can be used to study ME2's interaction with other proteins in mitochondrial function?

To investigate ME2's protein interactions and role in mitochondrial networks:

• Co-immunoprecipitation (Co-IP): Use validated ME2 antibodies (such as those tested in HeLa cells) to pull down ME2 complexes and identify interacting partners by mass spectrometry

• Proximity labeling: Combine ME2 antibodies with BioID or APEX2 approaches to identify proximal proteins in the mitochondrial environment

• Confocal microscopy with co-localization: Utilize ME2 antibodies validated for immunofluorescence to visualize spatial relationships with other mitochondrial proteins

• FRET/FLIM assays: Employ fluorophore-conjugated ME2 antibodies to detect direct protein interactions in live or fixed cells

When designing these experiments, remember that ME2 localizes to the "mitochondrion matrix" and consider how this compartmentalization affects experimental design and interpretation.

How can different ME2 antibody conjugates be utilized in multiplexed imaging studies?

Multiplexed imaging using various ME2 antibody conjugates enables simultaneous visualization of multiple targets:

• Fluorophore selection: Multiple ME2 antibody conjugates are available, including:

  • FITC conjugated antibodies for green fluorescence

  • Alexa Fluor 647 conjugated antibodies for far-red detection

  • CoraFluor conjugated antibodies for specialized applications

• Multiplex strategy:

  • Combine ME2 antibodies with antibodies against other metabolic enzymes

  • Use antibodies from different host species (rabbit vs. mouse) with species-specific secondary antibodies

  • Employ directly conjugated antibodies to avoid secondary antibody cross-reactivity

• Sequential detection:

  • For highly complex panels, consider sequential detection with antibody stripping

  • Validate that stripping protocols do not affect ME2 epitope integrity

• Spatial analysis:

  • Pair ME2 detection with mitochondrial markers to assess potential changes in localization under different conditions

  • Correlate ME2 expression with functional mitochondrial parameters

For optimal results, consider antibodies specifically validated for immunofluorescence applications with clear subcellular localization data .

What is the evidence linking ME2 to neurological disorders, and how can antibodies help investigate this connection?

ME2 has been implicated in neurological conditions, particularly epilepsy. Research strategies using ME2 antibodies include:

• Expression analysis: Compare ME2 expression in patient vs. control brain tissue using IHC with antibodies validated for neural tissues

• Genetic correlation: Combine ME2 antibody-based protein expression studies with genetic data to understand how specific ME2 variants affect protein levels and function

• Functional studies: Investigate ME2's role in GABA synthesis using antibodies to track ME2 expression in GABAergic neurons

• Cellular models: Use ME2 antibodies to validate knockout/knockdown models of epilepsy-associated ME2 mutations

The ME2 gene has been specifically "linked to epilepsy syndromes" through both "case-control and family-based association methods" , providing a foundation for antibody-based investigation of mechanistic connections.

How can ME2 antibodies contribute to understanding the regulatory mechanisms of cellular metabolism?

ME2 antibodies can be powerful tools for dissecting metabolic regulatory networks:

• Transcriptional regulation: Investigate the reported "reciprocal regulation" of ME2 by p53, which "has been shown to modulate metabolism and senescence"

• Post-translational modifications: Develop and use antibodies specific to modified forms of ME2 to understand how PTMs affect enzyme activity

• Metabolic adaptation: Track ME2 expression changes during metabolic stress, hypoxia, or nutrient deprivation

• Comparative analysis: Study ME2 alongside related enzymes (ME1, ME3) to understand their coordinated regulation

ME2 is especially interesting in metabolic studies because "ME1, ME2 are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect" , suggesting unique regulatory mechanisms worthy of investigation.