AMMECR1 Antibody

What is AMMECR1 and what cellular functions does it perform?

AMMECR1 is an X-linked gene located in the Xq22.3–Xq23 region that has been implicated in a contiguous gene deletion syndrome known as AMME. This syndrome is characterized by Alport syndrome with intellectual disability (Mental retardation), Midface hypoplasia, and Elliptocytosis. The protein encoded by AMMECR1 appears to play a critical role in craniofacial development, hematopoiesis, and neurological function, though its precise molecular mechanisms remain under investigation. Recent research has demonstrated that a single point mutation in AMMECR1 (c.G530A; p.G177D) can cause midface hypoplasia, mild intellectual disability, and elliptocytosis, indicating its importance in multiple developmental pathways .

The gene is part of a region that also includes several other important genes such as COL4A5, GUCY2F, NXT2, KCNE1L, and others, with deletions in this region being associated with the AMME syndrome phenotype. Significantly, studies have shown that AMMECR1 mutations alone can produce several key features of this syndrome, highlighting its central role in the pathogenesis .

How does AMMECR1 protein localize within cells and what implications does this have for antibody selection?

AMMECR1 exhibits a specific nuclear localization pattern in wild-type cells. Research using GFP-tagged AMMECR1 vectors has demonstrated that the wild-type protein localizes to the nucleus in multiple cell lines, including HEK293, COS-7, and HeLa cells. Interestingly, mutations such as the p.G177D variant show aberrant nuclear localization patterns compared to the wild type, suggesting that proper nuclear localization is essential for normal AMMECR1 function .

When selecting antibodies for AMMECR1 research, this nuclear localization pattern serves as an important validation criterion. Researchers should verify that their chosen antibody correctly identifies the nuclear expression pattern in control samples. For experiments involving mutant AMMECR1, antibodies capable of detecting both normal and aberrant localization patterns are essential for accurate characterization of pathological mechanisms .

What applications are AMMECR1 antibodies validated for, and how should I choose between them?

AMMECR1 antibodies are validated for multiple applications including immunohistochemistry (IHC), immunocytochemistry-immunofluorescence (ICC-IF), and Western blotting (WB) . For detection and quantification of AMMECR1 in biological fluids and cell lysates, ELISA kits are also available with a detection range of 0.313-20 ng/ml and sensitivity below 0.19 ng/ml .

When selecting an appropriate antibody, consider the following application-specific factors:

The final selection should be based on your specific experimental needs, including species reactivity (human AMMECR1 antibodies may have varying cross-reactivity with other species), the specific epitope recognized (especially important when studying mutant forms), and the validation data available for your application of interest .

How can I validate the specificity of an AMMECR1 antibody for my research application?

Validating antibody specificity is critical for ensuring reliable experimental results. For AMMECR1 antibodies, a multi-faceted validation approach is recommended:

• Genetic validation: Compare antibody performance in wild-type cells versus those with AMMECR1 knockdown/knockout. A specific antibody will show significantly reduced or absent signal in genetic models lacking AMMECR1.

• Cellular localization assessment: AMMECR1 exhibits a characteristic nuclear localization pattern. Validate your antibody by confirming it produces the expected nuclear staining pattern in immunofluorescence experiments using known AMMECR1-expressing cell lines such as HEK293, COS-7, or HeLa cells .

• Western blot analysis: Confirm the antibody detects a protein of the expected molecular weight (approximately 35-40 kDa for human AMMECR1) with minimal non-specific bands.

• Mutation analysis: If possible, test the antibody against cells expressing wild-type versus mutant AMMECR1 (such as the p.G177D variant). This can reveal whether the antibody can detect both forms and potentially highlight differences in localization or expression patterns .

• Epitope specificity: For polyclonal antibodies like the rabbit anti-AMMECR1 antibody HPA051762, understanding the epitope region can help determine whether the antibody will recognize specific mutant forms of interest .

What is the optimal protocol for visualizing subcellular localization of wild-type versus mutant AMMECR1?

Based on published methodologies, the following protocol can be used to visualize and compare wild-type and mutant AMMECR1 localization:

• Cell culture preparation: Culture appropriate cell lines (HEK293, COS-7, or HeLa) on sterile coverslips in six-well plates using DMEM supplemented with 10% FCS .

• Transfection: Transiently transfect cells with 3 μg of either wild-type or mutant GFP-tagged AMMECR1 using an appropriate transfection reagent (e.g., jetPEI). Include negative controls (transfection reagent without DNA) .

• Incubation and fixation: After 4 hours, remove transfection complexes and replace with fresh media. Allow expression for 24-48 hours before fixing cells with 4% paraformaldehyde.

• Counterstaining and mounting: Counterstain nuclei with DAPI and mount coverslips using an appropriate mounting medium.

• Microscopy and analysis: Visualize using confocal microscopy and analyze the nuclear localization patterns, documenting any differences between wild-type and mutant proteins.

When analyzing results, wild-type AMMECR1 typically shows a distinct nuclear localization pattern, while mutants like p.G177D demonstrate aberrant patterns that may include cytoplasmic mislocalization or altered nuclear distribution .

How can I optimize AMMECR1 detection in patient samples for clinical research studies?

Optimizing AMMECR1 detection in patient samples requires careful consideration of sample preparation, antibody selection, and detection methods:

• Sample selection and preparation:

  • For blood samples: Consider using peripheral blood smears to detect elliptocytes, which may be present in patients with AMMECR1 mutations .

  • For tissue biopsies: Optimal fixation methods should be established (typically 10% neutral buffered formalin) with standardized fixation times.

• Antibody optimization:

  • Titrate antibody concentrations (starting with manufacturer recommendations, typically around 0.05 mg/ml for polyclonal antibodies) .

  • Perform antigen retrieval optimization if using fixed tissues (testing both heat-induced and enzymatic methods).

  • Validate antibody performance on known positive and negative control tissues.

• Detection methods:

  • For protein quantification: ELISA assays offer sensitivity below 0.19 ng/ml .

  • For localization studies: Immunohistochemistry or immunofluorescence with careful attention to background reduction.

  • For mutation-specific detection: Consider using antibodies raised against specific regions containing known mutations if available.

• Controls and standardization:

  • Include samples from healthy controls alongside patient samples.

  • Use standardized protocols and automated systems where possible to reduce inter-assay variability.

  • Implement blinded analysis to prevent observer bias.

How can I effectively study the functional impact of AMMECR1 mutations using antibody-based approaches?

To effectively study the functional impact of AMMECR1 mutations using antibody-based approaches, consider implementing the following strategies:

• Site-directed mutagenesis and expression:

  • Generate mutant constructs of interest (such as the p.G177D variant) using site-directed mutagenesis on wild-type AMMECR1 vectors .

  • Design mutagenesis primers containing the specific point mutation (e.g., G>A mutation for p.G177D).

  • Perform PCR mutagenesis using high-fidelity polymerases like Pfu Ultra DNA polymerase.

  • Confirm mutations by sequencing before proceeding with expression studies.

• Comparative localization studies:

  • Transfect wild-type and mutant constructs into appropriate cell lines.

  • Use antibodies validated for immunofluorescence to compare localization patterns.

  • Quantify differences in nuclear versus cytoplasmic distribution using image analysis software.

• Protein-protein interaction analysis:

  • Employ co-immunoprecipitation with AMMECR1 antibodies to identify differential binding partners between wild-type and mutant proteins.

  • Use proximity ligation assays to visualize and quantify protein interactions in situ.

• Functional rescue experiments:

  • In cells with AMMECR1 knockdown, compare the ability of wild-type versus mutant AMMECR1 to rescue the phenotype.

  • Use antibodies to verify expression levels and localization in rescue experiments.

• Patient-derived materials:

  • When available, examine blood films for the presence of elliptocytes in patients with AMMECR1 mutations .

  • Compare AMMECR1 expression and localization in patient-derived cells versus healthy controls.

What are the key considerations when interpreting contradictory results from different AMMECR1 antibodies?

When faced with contradictory results from different AMMECR1 antibodies, researchers should systematically evaluate several key factors:

• Epitope differences:

  • Different antibodies may recognize distinct epitopes within AMMECR1, which can lead to varying results, especially if certain epitopes are masked in specific cellular contexts or protein complexes.

  • Map the epitopes recognized by each antibody and determine if your mutation or experimental conditions might affect epitope accessibility.

• Antibody validation status:

  • Assess the validation data for each antibody, including specificity testing and performance in applications similar to yours.

  • Polyclonal antibodies like those from Atlas Antibodies undergo standardized validation for IHC, ICC-IF, and WB applications .

• Technical variables:

  • Differences in sample preparation, fixation methods, antigen retrieval techniques, and detection systems can all contribute to discrepant results.

  • Systematically test different protocol parameters to determine if technical factors explain the contradictions.

• Isotype and host species considerations:

  • Different antibody isotypes and host species may exhibit varying levels of background binding or non-specific interactions.

  • Compare the performance of antibodies from different species or isotypes under identical conditions.

• Resolution approach:

  • Implement orthogonal validation methods that don't rely on antibodies (e.g., mRNA analysis, tagged protein expression).

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to confirm specificity.

  • Consider performing epitope competition assays to determine if antibodies recognize the same or different regions of AMMECR1.

What are the best methods for quantifying AMMECR1 expression levels in research samples?

Accurate quantification of AMMECR1 expression requires selecting appropriate methods based on your research questions and sample types:

• ELISA-based quantification:

  • Sandwich ELISA kits specifically designed for AMMECR1 offer a quantitative approach with high sensitivity (< 0.19 ng/ml) and a test range of 0.313-20 ng/ml .

  • Optimal for tissue homogenates, cell lysates, and biological fluids.

  • Provides absolute quantification through comparison with a standard curve.

• Western blot quantification:

  • Appropriate for relative quantification across samples.

  • Requires normalization to housekeeping proteins.

  • Enables detection of specific AMMECR1 variants or post-translational modifications.

  • Validate antibody specificity through appropriate controls.

• qRT-PCR for mRNA expression:

  • Complements protein-level measurements.

  • Useful for examining transcriptional regulation of AMMECR1.

  • Important to validate with protein expression data due to potential post-transcriptional regulation.

• Immunohistochemistry/immunofluorescence quantification:

  • Enables spatial analysis of expression within tissues or cells.

  • Can be semi-quantitative through image analysis software.

  • Important to establish standardized acquisition parameters and analysis methods.

• Flow cytometry:

  • Useful for analyzing AMMECR1 expression in mixed cell populations.

  • Requires permeabilization protocols for intracellular/nuclear antigens.

  • Enables correlation with other cellular markers.

How should I approach troubleshooting non-specific binding when using AMMECR1 antibodies?

Non-specific binding is a common challenge in antibody-based applications. For AMMECR1 antibodies, consider the following systematic troubleshooting approach:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers).

  • Increase blocking time or concentration if background remains high.

  • Consider adding protein (0.1-0.5% BSA) to antibody dilution buffers.

• Antibody dilution optimization:

  • Perform a dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

  • For polyclonal AMMECR1 antibodies (typically provided at 0.05 mg/ml), start with the manufacturer's recommended dilution and adjust as needed .

• Washing protocol refinement:

  • Increase washing duration or number of washes.

  • Add detergents (0.1-0.5% Tween-20) to wash buffers to reduce hydrophobic interactions.

  • Consider automated washing systems for consistent results.

• Cross-reactivity assessment:

  • Test the antibody on samples known to be negative for AMMECR1.

  • For immunoprecipitation experiments, include pre-clearing steps to remove non-specific binding proteins.

  • Consider using monoclonal antibodies if available, which may offer higher specificity.

• Fixation and antigen retrieval optimization:

  • For IHC/IF applications, compare different fixation methods.

  • Test various antigen retrieval protocols (pH ranges, heat-induced vs. enzymatic).

  • Optimize incubation times and temperatures.

• Detection system considerations:

  • Compare direct vs. indirect detection methods.

  • Test alternative secondary antibodies if using indirect detection.

  • For fluorescent applications, include appropriate controls for autofluorescence.