METAP1 Antibody

What is the biological function of METAP1 in cellular processes?

METAP1 (Methionine Aminopeptidase 1) is a member of the M24 family of metalloproteases that catalyzes the removal of the initiator methionine residue from nascent peptides during protein synthesis. This post-translational modification is essential for proper protein maturation and cellular function . METAP1 plays a particularly crucial role in cell cycle regulation, specifically during the G2/M phase transition, making it essential for normal cell cycle progression and cellular proliferation . Research has demonstrated that when METAP1 activity is inhibited or the protein is knocked down using gene-specific siRNA, cells exhibit delayed progression through the G2/M phase, highlighting its importance in cell division processes .

How does METAP1 differ structurally and functionally from other methionine aminopeptidases?

METAP1 belongs to the same enzymatic family as METAP2, but possesses distinct structural and functional characteristics. The purified recombinant human METAP1 protein typically consists of amino acid residues 52/53 to 386, with research showing that the N-terminal 89 amino acid region is not essential for its catalytic activity . Unlike METAP2, which when inhibited causes cell cycle arrest at the G1/S phase, METAP1 inhibition results in G2/M phase delay . This functional differentiation is further evidenced by experiments showing that overexpression of METAP1, but not METAP2, confers resistance to METAP1-specific inhibitors, indicating non-redundant roles in cellular processes .

What are the known protein substrates of METAP1 in mammalian cells?

One well-characterized substrate of METAP1 in mammalian cells is the 14-3-3γ protein. When METAP1 activity is inhibited, researchers observe increased retention of the N-terminal methionine of the 14-3-3γ protein, which can be detected using methionylated N-terminal fragment-specific antibodies . This molecular marker serves as an effective readout for assessing METAP1 inhibition in cellular assays. The relationship between METAP1 and its substrates is particularly relevant in understanding cell cycle regulation, as proper processing of these target proteins appears to be necessary for normal G2/M phase progression .

What are the optimal experimental conditions for detecting METAP1 using Western blot?

For optimal Western blot detection of METAP1 (MW: 43215 Da), researchers should follow these methodological guidelines:

• Sample preparation: Use standard cell lysis buffers containing protease inhibitors to prevent degradation of METAP1.

• Gel electrophoresis: Separate proteins using 10-12% SDS-PAGE gels.

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes using standard transfer conditions.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST.

• Primary antibody: Incubate with anti-METAP1 antibody at the recommended dilution (1:4000 for monoclonal antibodies or as specified by the manufacturer).

• Detection: Use appropriate secondary antibodies and detection systems.

It's important to note that multiple antibody options exist, including mouse monoclonal (clone 248CT14.6.1) and polyclonal antibodies, each with specific reactivity profiles primarily against human METAP1 .

How can METAP1 antibodies be effectively used in immunoprecipitation experiments?

For effective immunoprecipitation (IP) of METAP1:

• Cell lysate preparation: Prepare cell lysates under non-denaturing conditions to preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein G beads to reduce non-specific binding.

• Antibody binding: Incubate cleared lysates with METAP1-specific antibodies (monoclonal antibodies are generally preferred for IP due to their specificity) .

• Precipitation: Add protein G beads to capture antibody-antigen complexes.

• Washing: Perform stringent washing steps to reduce background.

• Elution: Elute precipitated proteins for downstream analysis.

This approach is particularly valuable for studying METAP1 interaction partners and post-translational modifications that may regulate its enzymatic activity or subcellular localization during different cell cycle phases.

What considerations should be made when selecting between monoclonal and polyclonal METAP1 antibodies?

The choice between monoclonal and polyclonal METAP1 antibodies should be guided by the specific experimental requirements:

When studying METAP1's role in cell cycle regulation, monoclonal antibodies may provide more consistent results for precise quantification, while polyclonal antibodies might be advantageous for detecting METAP1 in fixed tissue samples where epitope accessibility could be limited.

How can METAP1 antibodies be used to investigate its role in cancer cell proliferation?

Researchers investigating METAP1's role in cancer can employ antibody-based approaches through several methodological strategies:

• Expression analysis: Use METAP1 antibodies for Western blot or immunohistochemistry to compare expression levels between normal and cancer tissues/cell lines.

• Knockdown validation: Confirm METAP1 knockdown efficiency after siRNA treatment using antibody-based detection methods .

• Inhibitor studies: Validate the specificity of METAP1 inhibitors by assessing substrate processing (e.g., 14-3-3γ N-terminal methionine retention) using specific antibodies .

• Resistance mechanisms: In cells overexpressing METAP1, use antibodies to quantify expression levels and correlate with sensitivity to inhibitors .

• Cell cycle analysis: Combine METAP1 antibody staining with flow cytometry to examine its expression levels during different cell cycle phases.

These approaches have revealed that METAP1 inhibition leads to G2/M phase accumulation and can induce apoptosis in leukemia cell lines, suggesting its potential as a therapeutic target for cancer treatment .

What are the technical challenges in detecting post-translational modifications of METAP1?

Detecting post-translational modifications (PTMs) of METAP1 presents several technical challenges:

• Epitope masking: PTMs may alter antibody recognition sites, necessitating careful antibody selection.

• Modification-specific antibodies: Currently, few antibodies specifically target modified forms of METAP1.

• Low abundance: Modified forms of METAP1 may exist at low concentrations, requiring enrichment techniques.

• Transient modifications: Some PTMs may be short-lived and difficult to capture without inhibitors of modification-removing enzymes.

To overcome these challenges, researchers should consider:

• Using phospho-specific antibodies if studying METAP1 phosphorylation

• Combining immunoprecipitation with mass spectrometry for unbiased PTM detection

• Employing PTM-preserving lysis buffers containing appropriate inhibitors

• Using PTM-enrichment techniques prior to antibody-based detection

Understanding METAP1 regulation through PTMs may provide insights into its cell cycle-dependent functions and potential dysregulation in disease states.

How can researchers validate the specificity of METAP1 antibodies in complex experimental systems?

Validating METAP1 antibody specificity is crucial for generating reliable data. Recommended validation approaches include:

• Genetic controls: Compare antibody reactivity in wild-type versus METAP1 knockout or knockdown cells to confirm specificity .

• Overexpression controls: Test antibody in cells overexpressing METAP1 to verify increased signal intensity .

• Peptide competition: Pre-incubate antibody with purified METAP1 protein or immunizing peptide to block specific binding.

• Multiple antibody comparison: Validate findings using different antibodies targeting distinct METAP1 epitopes .

• Orthogonal techniques: Correlate antibody-based detection with mRNA expression or activity-based assays.

For the mouse monoclonal antibody (clone 248CT14.6.1), validation has confirmed specificity for human METAP1 in Western blot applications, while polyclonal antibodies have been validated for both Western blot and ELISA techniques .

What are the best preservation methods for maintaining METAP1 antibody activity during storage?

To maintain optimal METAP1 antibody activity during storage:

• Aliquoting: Divide antibody solutions into small, single-use aliquots to minimize freeze-thaw cycles.

• Storage temperature: Store at -20°C or -80°C as recommended by manufacturers .

• Freeze-thaw cycles: Strictly avoid repeated freeze-thaw cycles that can lead to protein denaturation .

• Buffer composition: Most METAP1 antibodies are supplied in PBS (pH 7.4) with 0.09% sodium azide as a preservative .

• Working dilutions: For short-term use (1-2 weeks), store diluted antibody at 4°C.

Following these guidelines is particularly important for ensuring consistent results in longitudinal studies examining METAP1's role in cell cycle regulation or cancer biology, where experimental variability must be minimized.

How can researchers optimize immunofluorescence protocols for subcellular localization studies of METAP1?

For optimal subcellular localization of METAP1 using immunofluorescence:

• Fixation: Test both paraformaldehyde (4%) and methanol fixation methods, as each may reveal different aspects of METAP1 localization.

• Permeabilization: Use 0.1-0.5% Triton X-100 or 0.1% saponin to access intracellular METAP1.

• Blocking: Block with 5% normal serum from the same species as the secondary antibody.

• Antibody dilution: Begin with the manufacturer's recommended dilution (e.g., 1:100-1:500) and optimize as needed.

• Controls: Include negative controls (secondary antibody only) and positive controls (cells known to express METAP1).

• Co-staining: Consider co-staining with markers for specific organelles to precisely determine METAP1 subcellular localization.

This approach can reveal METAP1's dynamic localization during different cell cycle phases, potentially providing insights into its functional role in G2/M progression as indicated by previous research .

What strategies can address non-specific binding issues when using METAP1 antibodies?

To minimize non-specific binding when using METAP1 antibodies:

• Blocking optimization: Test different blocking agents (BSA, normal serum, commercial blockers) to identify the most effective option.

• Antibody dilution: Use the optimal antibody dilution; for monoclonal METAP1 antibodies, 1:4000 has been recommended for Western blot applications .

• Washing stringency: Increase the number and duration of washing steps with detergent-containing buffers.

• Pre-adsorption: For tissues with high background, pre-adsorb antibodies with tissue powder from the species being studied.

• Secondary antibody selection: Choose highly cross-adsorbed secondary antibodies to reduce cross-reactivity.

• Buffer additives: Consider adding 0.1-0.5% Tween-20 or Triton X-100 to reduce hydrophobic interactions.

Addressing non-specific binding is particularly important when studying METAP1 in complex tissue samples or when trying to detect subtle changes in expression or localization during cell cycle progression or in response to inhibitors .

How should researchers interpret variations in METAP1 detection across different cell lines or tissue samples?

When interpreting variations in METAP1 detection across samples:

• Expression normalization: Always normalize METAP1 signals to appropriate loading controls (e.g., β-actin, GAPDH) for quantitative comparisons.

• Cell cycle considerations: Since METAP1 plays a role in G2/M phase progression , variations may reflect differences in cell cycle distribution rather than actual expression differences.

• Antibody validation: Confirm that the antibody performs consistently across the cell lines or tissues being compared.

• Post-translational modifications: Consider that variations might reflect differences in METAP1 modification status rather than total protein levels.

• Isoform expression: Be aware that different isoforms or splice variants may be expressed in different tissues.

Understanding these factors is crucial when studying METAP1's potential role in cancer, where altered expression or activity might contribute to dysregulated cell proliferation .

What are the key considerations when analyzing METAP1 expression in relation to cell cycle phases?

When analyzing METAP1 in relation to cell cycle phases:

• Synchronization methods: Consider how different cell synchronization techniques might affect METAP1 expression or activity.

• Cell cycle markers: Co-stain for established cell cycle phase markers (e.g., phospho-histone H3 for mitosis) to correlate METAP1 patterns with specific phases.

• Time-course experiments: Design time-course experiments after synchronization release to track METAP1 dynamics throughout the cell cycle.

• Inhibitor effects: When using METAP1 inhibitors, carefully distinguish between direct effects on METAP1 and secondary effects on the cell cycle.

• Single-cell analysis: Consider flow cytometry or immunofluorescence approaches for analyzing METAP1 at the single-cell level across different cell cycle phases.

Research has established that METAP1 inhibition or knockdown leads to G2/M phase delay, suggesting a critical role in this transition point that requires careful experimental design to fully characterize .

How can researchers accurately quantify METAP1 enzyme activity in relation to antibody-detected protein levels?

To correlate METAP1 protein levels with enzymatic activity:

• Activity assays: Implement methionine aminopeptidase activity assays using fluorogenic or chromogenic substrates.

• Substrate processing: Monitor the processing status of known METAP1 substrates, such as 14-3-3γ protein, using antibodies that specifically recognize the methionylated N-terminal fragment .

• Inhibitor controls: Include METAP1-specific inhibitors as controls to establish baseline activity levels.

• Correlation analysis: Perform statistical correlation analyses between antibody-detected protein levels and enzymatic activity measurements.

• Recombinant protein standards: Use purified recombinant METAP1 protein to establish standard curves for both antibody detection and activity assays.

This integrated approach provides more comprehensive insights than protein detection alone, particularly when studying the functional consequences of METAP1 inhibition in cancer research or cell cycle studies .