metap1d Antibody

What is METAP1D and what is its biological significance?

METAP1D (Methionine Aminopeptidase 1D, Mitochondrial) is an enzyme that removes the N-terminal methionine from nascent proteins. This process is essential when the second residue in the protein sequence is small and uncharged (Met-Ala-, Cys, Gly, Pro, Ser, Thr, or Val). METAP1D specifically requires deformylation of the N(alpha)-formylated initiator methionine before hydrolysis can occur. Importantly, METAP1D has been found to be overexpressed in colon cancer cell lines and colon tumors compared to normal tissues at the protein level, suggesting it may play a significant role in colon tumorigenesis . As a mitochondrially localized protein, it represents an important target for understanding mitochondrial protein processing and potential cancer-related mechanisms .

What types of METAP1D antibodies are currently available for research applications?

Currently, there are several types of METAP1D antibodies available for research, primarily rabbit polyclonal antibodies. These antibodies have been validated for various applications including Western Blotting (WB), Immunohistochemistry (IHC), and ELISA . The antibodies are typically generated using recombinant human METAP1D protein segments as immunogens. For example, some are produced using E.coli-derived human METAP1D recombinant protein (Position: Q36-A335) , while others utilize fusion proteins of human METAP1D . Most of these antibodies demonstrate cross-reactivity with human, mouse, and rat METAP1D proteins, making them versatile for comparative studies across species .

How are METAP1D antibodies validated for research applications?

METAP1D antibodies undergo rigorous validation processes to ensure specificity and reproducibility. Validation typically involves:

• Western blot analysis across multiple cell lines to confirm target specificity and molecular weight recognition (approximately 37 kDa for METAP1D)

• Immunohistochemistry on paraffin-embedded tissue sections, particularly colorectal adenocarcinoma tissue where METAP1D is known to be overexpressed

• Cross-reactivity testing against human, mouse, and rat samples to ensure species compatibility

• Performance assessment in different applications (WB, IHC, ELISA) with standardized protocols

Premium antibodies (such as those labeled as "Picoband") undergo additional validation to ensure high affinity and signal-to-noise ratio, particularly important for detecting proteins that may be expressed at lower levels .

What are the optimal conditions for using METAP1D antibodies in Western blot applications?

For optimal Western blot results with METAP1D antibodies, the following conditions have been empirically determined:

These conditions have been validated across multiple cell lines including human HEL, K562, 293T, HepG2, THP-1, and HeLa cells, as well as rat liver tissue, rat RH35 cells, and mouse Neuro-2a cells . It's important to note that METAP1D appears most consistently at its calculated molecular weight of 37.262 kDa across different sample types .

What antigen retrieval method is most effective for METAP1D immunohistochemistry?

For effective METAP1D detection in immunohistochemistry applications, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has proven most effective . The complete recommended protocol includes:

• Deparaffinization and rehydration of paraffin-embedded tissue sections

• Heat-mediated antigen retrieval using EDTA buffer (pH 8.0)

• Blocking with 10% goat serum to reduce non-specific binding

• Incubation with rabbit anti-METAP1D antibody at 2 μg/ml concentration overnight at 4°C

• Application of peroxidase-conjugated goat anti-rabbit IgG as secondary antibody (30 minutes incubation at 37°C)

• Development using DAB (3,3'-diaminobenzidine) as the chromogen

This protocol has been validated particularly on human colorectal adenocarcinoma tissue, where METAP1D is known to be upregulated compared to normal tissue .

How should METAP1D antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of METAP1D antibodies is critical for maintaining their activity and ensuring reproducible results:

• Long-term storage: Lyophilized antibodies should be stored at -20°C for up to one year from the date of receipt .

• After reconstitution:

  • Store at 4°C for up to one month for immediate use

  • For longer storage, aliquot and freeze at -20°C for up to six months

  • Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Reconstitution method: Add 0.2 ml of distilled water to lyophilized antibody to yield a concentration of 500 μg/ml .

• Buffer composition: Reconstituted antibodies typically contain buffer components that stabilize the antibody (e.g., 4 mg Trehalose, 0.9 mg NaCl, 0.2 mg Na2HPO4 per vial) .

Following these storage guidelines helps ensure consistent antibody performance across experiments and over time.

How can researchers distinguish between specific and non-specific binding when using METAP1D antibodies?

Distinguishing between specific and non-specific binding is critical for accurate interpretation of METAP1D antibody results:

• Molecular weight verification: METAP1D should appear at approximately 37 kDa in Western blot applications. Bands at significantly different molecular weights may indicate non-specific binding .

• Positive and negative controls:

  • Use cell lines known to express METAP1D (e.g., colon cancer cell lines) as positive controls

  • Include normal tissues with lower expression as comparative controls

  • Consider using METAP1D knockdown or knockout samples when available

• Cross-validation: Compare results across multiple detection methods (WB, IHC, ELISA) to confirm target specificity .

• Blocking peptide competition: If available, pre-incubate the antibody with a blocking peptide containing the immunogen sequence. Specific signals should be significantly reduced or eliminated after this competition .

• Signal localization: In IHC applications, METAP1D should primarily localize to mitochondria. Diffuse or predominantly nuclear staining patterns may indicate non-specific binding .

These approaches collectively provide confidence in the specificity of detected signals and help identify potential artifacts.

What are common pitfalls in METAP1D antibody applications and how can they be addressed?

Several common pitfalls can affect METAP1D antibody experiments, each with specific solutions:

Addressing these common issues systematically can significantly improve experimental outcomes when working with METAP1D antibodies.

How should researchers interpret conflicting results between different METAP1D antibody applications?

When faced with conflicting results between different METAP1D antibody applications (e.g., discrepancies between WB and IHC results), researchers should consider:

• Epitope accessibility: The 3D conformation of METAP1D may differ between applications, affecting epitope exposure. Western blot uses denatured proteins, while IHC may preserve some structural elements that can mask epitopes .

• Expression threshold detection: Western blot and ELISA may have different sensitivity thresholds compared to IHC, leading to apparent discrepancies in expression levels .

• Spatial resolution considerations: IHC provides spatial information about protein localization, while WB gives information about total protein abundance. METAP1D's mitochondrial localization may create apparent discrepancies between these techniques .

• Verification strategy: When encountering discrepancies, employ orthogonal techniques:

  • Use antibodies targeting different epitopes

  • Complement antibody-based detection with mRNA expression analysis

  • Consider mass spectrometry-based proteomics for definitive identification

  • Use cellular fractionation to verify mitochondrial localization

• Technical validation: Review controls for each method and consider repeating experiments with optimized conditions specific to each application .

By systematically evaluating these factors, researchers can better interpret and reconcile seemingly conflicting results.

How can METAP1D antibodies be effectively used to study its role in cancer progression?

Given METAP1D's overexpression in colon cancer cell lines and tumors , researchers can employ several strategic approaches to study its role in cancer progression:

• Comparative expression analysis: Use validated METAP1D antibodies to:

  • Compare expression levels between matched tumor and adjacent normal tissues

  • Analyze expression across cancer progression stages (adenoma to carcinoma)

  • Correlate expression with clinical outcomes in patient cohorts

• Co-localization studies: Combine METAP1D antibodies with mitochondrial markers to:

  • Assess changes in mitochondrial localization during cancer progression

  • Investigate relationships with mitochondrial dysfunction markers

• Functional validation: Combine antibody detection with:

  • siRNA or CRISPR knockout of METAP1D followed by phenotypic assessment

  • Rescue experiments in knockout models

  • Drug inhibition studies targeting METAP1D enzymatic activity

• Signaling pathway analysis: Use METAP1D antibodies in combination with:

  • Phospho-specific antibodies for related signaling pathways

  • Co-immunoprecipitation to identify METAP1D interaction partners in cancer cells

  • Chromatin immunoprecipitation (if nuclear localization is observed) to identify potential gene regulatory roles

These approaches can provide comprehensive insights into METAP1D's functional significance in cancer contexts.

What methodologies can be used to study METAP1D's role in protein N-terminal methionine processing?

To investigate METAP1D's enzymatic function in N-terminal methionine processing, researchers can implement several methodologies:

• In vitro enzymatic assays:

  • Use immunoprecipitated METAP1D (using validated antibodies) to assess enzymatic activity on synthetic peptide substrates

  • Monitor cleavage of fluorogenic substrates to measure kinetic parameters

  • Compare wild-type and mutant METAP1D activity to identify critical residues

• Substrate identification:

  • Combine METAP1D knockdown/overexpression with N-terminal proteomics

  • Use stable isotope labeling by amino acids in cell culture (SILAC) to quantitatively compare N-terminal peptides in control versus METAP1D-manipulated samples

  • Validate findings using Western blotting with METAP1D antibodies and substrate-specific antibodies

• Mitochondrial import studies:

  • Use METAP1D antibodies to track protein localization during mitochondrial protein import

  • Perform subcellular fractionation followed by Western blot to confirm mitochondrial localization

  • Combine with pulse-chase experiments to track processing kinetics

• Structural studies:

  • Use purified METAP1D (validated by antibody recognition) for crystallography studies

  • Perform hydrogen-deuterium exchange mass spectrometry with and without substrates

  • Develop structure-based inhibitors and test their efficacy using antibody-based detection methods

These methodologies collectively provide a comprehensive understanding of METAP1D's enzymatic function and substrate specificity.

How can deamidation prediction models inform the design of experiments using METAP1D antibodies?

Recent advances in deamidation prediction models, as described in search result , provide important considerations for METAP1D antibody-based experiments:

• Epitope stability assessment:

  • Use deamidation prediction models to identify potential deamidation sites within METAP1D

  • Select antibodies whose epitopes avoid regions prone to deamidation

  • For critical experiments, consider multiple antibodies targeting distinct epitopes with different deamidation susceptibilities

• Sample preparation optimization:

  • Accelerated thermal stress (e.g., 40°C incubation) can induce deamidation

  • When comparing samples, ensure consistent sample preparation conditions to avoid deamidation-related artifacts

  • Include appropriate controls to account for potential deamidation during sample processing

• Integration with peptide mapping:

  • Modern peptide mapping techniques allow site-specific identification of deamidation sites

  • Combine antibody-based detection with peptide mapping to correlate functional changes with specific modification sites

  • Implement high-throughput peptide mapping protocols to increase experimental throughput

• Experimental design considerations:

  • When studying METAP1D in long-term storage or stress conditions, account for potential deamidation effects

  • Consider how buffer conditions (particularly pH and temperature) might influence deamidation rates

  • Implement supervised machine learning approaches to predict deamidation propensities throughout METAP1D sequence

By incorporating these deamidation prediction insights, researchers can design more robust experiments and better interpret results from METAP1D antibody-based studies.

How might METAP1D antibodies be utilized in emerging single-cell and spatial proteomics applications?

METAP1D antibodies can be adapted for cutting-edge single-cell and spatial proteomics applications through several innovative approaches:

• Single-cell proteomics:

  • Adapt METAP1D antibodies for mass cytometry (CyTOF) by metal conjugation

  • Utilize antibodies in microfluidic-based single-cell Western blotting

  • Implement METAP1D detection in single-cell proteomic workflows to correlate with transcriptomic data

• Spatial proteomics applications:

  • Optimize METAP1D antibodies for multiplexed immunofluorescence techniques

  • Implement cyclic immunofluorescence (CycIF) to co-localize METAP1D with multiple mitochondrial and cancer markers

  • Adapt antibodies for emerging spatial transcriptomics-proteomics integrated platforms

• In situ proximity labeling:

  • Develop METAP1D antibody-based proximity labeling reagents

  • Use to identify spatial interaction partners in intact cellular environments

  • Combine with mitochondrial markers to map METAP1D's local environment

These emerging technologies will provide unprecedented insights into METAP1D's function at single-cell resolution and within complex tissue architectures.

What considerations are important when designing METAP1D antibody-based diagnostic assays for cancer detection?

When developing METAP1D antibody-based diagnostic assays for potential cancer applications, researchers should consider:

• Antibody pair selection:

  • Identify non-competing antibody pairs that recognize distinct METAP1D epitopes

  • Evaluate capture and detection antibody combinations for optimal sensitivity and specificity

  • Consider the stability of selected epitopes under various sample processing conditions

• Assay platform optimization:

  • Compare performance across platforms (ELISA, multiplex bead arrays, automated immunoassays)

  • Determine minimum detection thresholds needed for clinical relevance

  • Develop calibration standards using recombinant METAP1D proteins

• Clinical validation strategy:

  • Define appropriate sample types (tissue, serum, circulating tumor cells)

  • Establish reference ranges across healthy and disease populations

  • Determine diagnostic accuracy metrics (sensitivity, specificity, positive/negative predictive values)

• Technical validation considerations:

  • Assess assay reproducibility across different laboratories

  • Establish standardized protocols to minimize pre-analytical variables

  • Determine stability of METAP1D in clinical samples under various storage conditions

These considerations are essential for translating basic METAP1D research into clinically relevant diagnostic applications.