AMFR Antibody

What is AMFR and what is its significance in cellular research?

AMFR (Autocrine Motility Factor Receptor) is a 73-75 kDa transmembrane glycoprotein that functions as an E3 ubiquitin-protein ligase. It plays critical roles in endoplasmic reticulum-associated degradation (ERAD), protein homeostasis maintenance, cell motility regulation, and lipid metabolism. The protein contains a RING finger domain essential for its ubiquitin ligase activity, which has led to its alternative designation as RNF45 (RING finger protein 45). AMFR's altered expression has been documented in various pathological conditions, including a notable decrease in plasma levels of osteoporosis patients, suggesting potential biomarker applications . AMFR has also been implicated in cancer metastasis, making it a significant target for oncology research.

What are the molecular characteristics of AMFR that impact antibody design and selection?

AMFR is characterized by:

• Molecular weight: 73-75 kDa (calculated/observed on SDS-PAGE)

• UniProt accession: Q9UKV5

• Multiple isoforms: At least two documented isoforms exist (isoform 1 and isoform 2)

• Transmembrane structure: Contains a transmembrane domain affecting epitope accessibility

• Post-translational modifications: May impact antibody recognition

The protein's structure includes specific domains that serve as optimal targets for antibody generation. Western blot analyses have shown that anti-AMFR antibodies may detect two bands in the predicted molecular weight range, particularly in tissue lysates, while plasma samples may show a single band between these two positions . This band pattern reflects the protein's isoforms and potential post-translational modifications that researchers must consider when selecting appropriate antibodies.

What species reactivity should researchers consider when selecting AMFR antibodies?

When selecting AMFR antibodies, researchers should consider the following species reactivity information:

Species cross-reactivity is critical for comparative studies and model organism research. The commercial antibody #9590 from Cell Signaling Technology has confirmed reactivity with human, monkey, and dog samples . Meanwhile, the DF7718 antibody has a broader predicted reactivity profile, including various mammalian species, though these predictions require validation for specific research applications . Sequence conservation analysis should be performed when working with species not listed in the documented reactivity profiles.

What are the recommended dilutions and applications for AMFR antibodies?

The following application-specific dilutions are recommended based on antibody validation data:

*Note: The optimal dilutions for each experimental system should be determined empirically by the researcher.

For Western blotting applications, a 1:1000 dilution is generally appropriate for detecting endogenous levels of AMFR in cell and tissue lysates . Immunoprecipitation applications typically require higher antibody concentrations, with a 1:50 dilution recommended . These dilutions should be optimized based on sample type, protein abundance, and detection method. For novel applications or sample types, a dilution series experiment is recommended to determine optimal conditions.

How should samples be prepared for optimal AMFR detection in Western blotting?

For optimal AMFR detection in Western blotting:

• Sample Preparation:

  • For tissue samples: Homogenize in RIPA buffer containing protease inhibitors

  • For plasma samples: Deplete abundant proteins (albumin and IgG) using affinity methods

  • For cell lysates: Standard lysis buffers with protease inhibitors are generally sufficient

• Protein Loading:

  • Load 10-30 μg of total protein for cell/tissue lysates

  • For plasma samples, higher amounts (≥1000 ng) may be necessary for detection of endogenous AMFR

• Gel Selection:

  • Use 4-12% Bis-Tris gels for optimal resolution of the 73-75 kDa AMFR protein

• Transfer Conditions:

  • Transfer to PVDF membranes (0.45 μm pore size) for optimal protein binding

  • Transfer at 30V overnight at 4°C for large proteins like AMFR

• Blocking Conditions:

  • Block with 5% milk powder in TBS containing 0.5% Tween 20 at room temperature (23°C) for 1 hour

Researchers should include appropriate positive controls, such as AMFR-overexpressing cell lysates, and negative controls lacking AMFR expression vector, which have proven valuable for antibody validation .

What verification methods ensure AMFR antibody specificity?

Multiple complementary methods should be employed to verify AMFR antibody specificity:

• Western Blot Analysis:

  • Compare reactivity patterns between samples with varying AMFR expression

  • Validate using AMFR-overexpressing cell lysates versus negative control cells

  • Expect bands at approximately 73-75 kDa corresponding to the predicted molecular weight

• Peptide Epitope Mapping:

  • High-density peptide arrays with overlapping peptides (e.g., 12 amino acids with 11-residue overlap) covering the antigen region

  • Identifies specific binding epitopes and potential cross-reactive sequences

• Protein Microarray Analysis:

  • Test antibody against arrays containing thousands of protein fragments

  • The anti-AMFR antibody HPA029018 showed high selectivity, with AMFR antigen detection at 26,641 AU compared to a median of 238 AU across all other antigens

  • Only approximately 0.1% of non-target antigens showed any reactivity, with signals not exceeding 15% of the AMFR protein fragment signal

• Immunohistochemistry Controls:

  • Include tissues known to express or lack AMFR

  • Compare staining patterns with independent antibodies targeting different AMFR epitopes

These verification steps are crucial for ensuring experimental reliability and reproducibility, particularly for studies examining AMFR in pathological conditions.

Why might multiple bands appear when detecting AMFR in Western blots?

Multiple bands in AMFR Western blots may appear due to:

• Isoform Detection: AMFR has documented isoforms (isoform 1 and 2) , which can appear as distinct bands. The Human Protein Atlas portal demonstrated that HPA029018 detected two bands within the predicted molecular weight range for AMFR (73 kDa) .

• Post-translational Modifications: Glycosylation, phosphorylation, or ubiquitination can alter protein migration. AMFR functions as an E3 ubiquitin ligase and may be subject to auto-ubiquitination.

• Proteolytic Processing: Sample degradation during preparation may generate fragments. In plasma samples, a weak band was observed between the two bands seen in tissue lysates .

• Cross-reactivity: Although rare with well-validated antibodies, epitope similarity with other proteins may occur. The epitope mapping of anti-AMFR antibody HPA029018 identified the QHA motif, which was present in three other antigens (NUTM1, RFX6, and BRD4) .

• Sample Type Differences: Different sample types (tissues versus plasma) show distinct banding patterns. Western blot analysis showed protein bands in a similar molecular weight range between plasma pools and AMFR-overexpressed cell lines .

To address multiple bands, researchers should employ positive controls (AMFR-overexpressing cells), conduct parallel experiments with multiple anti-AMFR antibodies targeting different epitopes, and consider protein deglycosylation experiments to identify post-translational modification contributions.

How can researchers differentiate between AMFR isoforms in experimental settings?

Differentiating between AMFR isoforms requires several specialized approaches:

• Isoform-Specific Antibodies:

  • Design or select antibodies targeting unique sequences in specific isoforms

  • Validate using recombinant protein standards representing each isoform

• RT-PCR and qPCR Analysis:

  • Design primers spanning exon junctions specific to each isoform

  • Quantify isoform-specific mRNA expression to correlate with protein levels

• Mass Spectrometry:

  • Perform immunoprecipitation with anti-AMFR antibodies followed by LC-MS/MS

  • Identify unique peptides corresponding to different isoforms

• Recombinant Expression:

  • Generate cell lines expressing individual AMFR isoforms

  • Compare migration patterns with endogenous samples

• SDS-PAGE Optimization:

  • Use gradient gels (4-15%) to improve resolution between closely migrating isoforms

  • Extended electrophoresis times can enhance separation

The two documented AMFR isoforms (isoform 1 and isoform 2) may have distinct functional roles, making their differentiation particularly important for studies investigating AMFR's role in disease processes and cellular pathways.

What approaches can be used to study AMFR's role in osteoporosis and other diseases?

Recent studies have implicated AMFR in osteoporosis pathophysiology, with several methodological approaches available for investigation:

• Affinity Proteomics:

  • Use antibody bead arrays for multiplexed protein analysis in body fluids

  • Research has demonstrated decreased AMFR levels in plasma of osteoporosis patients using this approach

  • Starting with large antibody panels (4608 antibodies) and narrowing to targeted arrays (180 antibodies) allows comprehensive protein profiling

• Population-Based Studies:

  • Analyze AMFR levels in well-characterized patient cohorts

  • The differential profile of AMFR between osteoporosis patients and matched controls was discovered in independent population-based studies

• Animal Models:

  • Generate AMFR knockout or conditional knockout models

  • Assess bone density, structure, and formation/resorption markers

• Cellular Mechanisms:

  • Investigate AMFR's E3 ubiquitin ligase activity in osteoblasts and osteoclasts

  • Identify AMFR substrates relevant to bone metabolism using immunoprecipitation and mass spectrometry

• Clinical Correlation:

  • Correlate AMFR plasma levels with bone mineral density and fracture risk

  • Evaluate AMFR as a potential biomarker for diagnosis and monitoring of patient mobility within osteoporosis

These approaches can be adapted to study AMFR's role in other pathological conditions, such as cancer, where AMFR has been implicated in metastasis and tumor progression.

How can epitope masking issues be addressed when detecting AMFR in fixed tissues?

Epitope masking is a common challenge in AMFR detection in fixed tissues, particularly given its transmembrane nature. To address this:

• Antigen Retrieval Optimization:

  • Heat-induced epitope retrieval: Test multiple buffers (citrate pH 6.0, EDTA pH 8.0, Tris-EDTA pH 9.0)

  • Enzymatic retrieval: Try proteinase K or trypsin for exposing certain epitopes

  • Combination approaches: Sequential heat and enzymatic treatments

• Fixation Considerations:

  • Minimize fixation time with formalin to reduce excessive cross-linking

  • Consider alternative fixatives (zinc-based fixatives, alcohol-based fixatives)

  • For fresh tissues, employ frozen sections to avoid fixation-related epitope masking

• Detergent Enhancement:

  • Include non-ionic detergents (0.1-0.3% Triton X-100) in antibody diluent

  • For membrane proteins like AMFR, this facilitates antibody penetration

• Antibody Selection Based on Epitope Mapping:

  • Choose antibodies targeting epitopes less affected by fixation

  • The anti-AMFR antibody HPA029018 recognizes specific epitope motifs including QHA , which may be differently affected by fixation than other epitopes

• Signal Amplification:

  • Employ tyramide signal amplification systems

  • Use polymer-based detection systems for enhanced sensitivity

These approaches should be systematically tested and optimized for specific tissue types and experimental questions.

What are the latest innovations in using AMFR antibodies for clinical research?

Recent advances in AMFR antibody applications for clinical research include:

• Biomarker Development:

  • AMFR plasma levels have been identified as potentially decreased in osteoporosis patients

  • Further validation in additional study sets will determine the clinical value of AMFR as a biomarker for diagnosis and monitoring patient mobility

• Multiplex Proteomic Profiling:

  • Integration of anti-AMFR antibodies in multiplexed antibody bead arrays

  • Starting with 4608 antibodies and plasma samples from 22 women for untargeted screening, researchers identified 72 proteins for further analysis, including AMFR

  • Targeted bead arrays with 180 antibodies profiling 92 proteins facilitated discovery of differential profiles between osteoporosis patients and controls

• High-Throughput Validation Platforms:

  • Custom high-density peptide microarrays for epitope mapping

  • Protein microarrays containing over 13,000 unique antigens for antibody selectivity verification

• Correlative Multi-Omic Studies:

  • Integration of AMFR antibody-based protein detection with genomic and transcriptomic data

  • Correlation of AMFR levels with genetic risk factors for osteoporosis

• Therapeutic Target Validation:

  • Using specific AMFR antibodies to block protein function in experimental models

  • Potential for developing therapeutic antibodies targeting AMFR in disease contexts

These innovative approaches demonstrate the evolving role of AMFR antibodies beyond basic research into clinical applications, particularly in osteoporosis where AMFR may aid understanding of disease mechanisms and support diagnostic tools .

What controls should be included when using AMFR antibodies in experimental protocols?

A robust experimental design for AMFR antibody applications should include:

• Positive Controls:

  • AMFR-overexpressing cell lysates (e.g., commercially available overexpression systems)

  • Tissues with known high AMFR expression (tonsil and liver have shown strong reactivity)

  • Reference cell lines with documented AMFR expression (RT-4 cells, U-251 MG cells)

• Negative Controls:

  • Cell lines lacking AMFR vector expression

  • Primary antibody omission controls

  • Isotype-matched irrelevant antibodies

• Technical Controls:

  • Loading controls for Western blotting (housekeeping proteins)

  • Internal reference proteins for normalization in quantitative analyses

  • Gradient of protein concentrations (1000, 100, 10, and 1 ng) to establish detection limits

• Validation Controls:

  • Multiple anti-AMFR antibodies targeting different epitopes

  • Peptide competition assays using the immunizing peptide

  • siRNA or CRISPR knockdown of AMFR to confirm specificity

• Sample Processing Controls:

  • For plasma samples, include IgG/HSA-depleted human plasma

  • Process matched samples simultaneously to minimize technical variation

These controls help ensure experimental reliability and facilitate accurate interpretation of results, particularly in complex sample types like plasma where AMFR detection may be challenging.

How should researchers design experiments to study AMFR's E3 ubiquitin ligase activity?

To investigate AMFR's E3 ubiquitin ligase activity, researchers should consider these experimental design elements:

• In Vitro Ubiquitination Assays:

  • Components: Purified E1, E2 (UBC7/UBE2G2), recombinant AMFR, ubiquitin, ATP, and potential substrates

  • Controls: Reactions lacking individual components, especially ATP

  • Detection: Anti-ubiquitin Western blotting or mass spectrometry

• Substrate Identification:

  • Co-immunoprecipitation with anti-AMFR antibodies followed by mass spectrometry

  • Yeast two-hybrid screening for AMFR-interacting proteins

  • Proximity labeling approaches (BioID or APEX) with AMFR as the bait

• Domain Function Analysis:

  • Generate RING finger domain mutants (C356S and H361A) to abolish E3 ligase activity

  • Compare substrate degradation rates between wild-type and mutant AMFR

  • Examine the impact on cellular processes like ERAD

• Cellular Ubiquitination Studies:

  • Express HA-tagged ubiquitin and immunoprecipitate with anti-HA antibodies

  • Probe for AMFR substrates in the presence and absence of proteasome inhibitors

  • Use siRNA-mediated AMFR knockdown as a control

• Physiological Relevance:

  • Correlate E3 ligase activity with pathological conditions like osteoporosis

  • Investigate AMFR-mediated protein degradation in disease-relevant cell types

These approaches can illuminate AMFR's role in protein quality control and various cellular pathways, potentially revealing therapeutic targets for diseases where AMFR dysfunction contributes to pathology.

How are AMFR antibodies being used in cancer research?

AMFR antibodies are facilitating several important areas of cancer research:

• Biomarker Studies:

  • AMFR expression has been correlated with cancer progression and metastasis

  • Early studies have shown altered AMFR expression in various cancer types, making it a potential prognostic marker

  • Immunohistochemical analysis using anti-AMFR antibodies in cancer tissue microarrays

• Metastasis Mechanisms:

  • AMFR's role as the receptor for autocrine motility factor suggests involvement in tumor cell migration

  • Anti-AMFR antibodies enable tracking of receptor localization during epithelial-mesenchymal transition

  • Co-localization studies with cytoskeletal markers to understand migration dynamics

• Therapeutic Targeting:

  • Development of function-blocking antibodies targeting extracellular domains of AMFR

  • Potential for antibody-drug conjugates directed against AMFR-expressing tumor cells

  • Evaluation of anti-AMFR antibodies for cancer immunotherapy applications

• Resistance Mechanisms:

  • AMFR's role in ERAD may influence cancer cell resistance to ER stress-inducing therapies

  • Quantification of AMFR levels in therapy-resistant versus sensitive tumor populations

  • Correlation with other ER stress response markers

• Patient Stratification:

  • Development of diagnostic panels including AMFR antibodies for patient subtyping

  • Correlation of AMFR expression patterns with treatment outcomes and survival

These applications highlight AMFR's emerging significance in cancer biology and the crucial role of well-characterized antibodies in advancing this research field.

What are the challenges in developing highly specific monoclonal antibodies against AMFR?

Developing highly specific monoclonal antibodies against AMFR faces several technical challenges:

• Epitope Selection Complexities:

  • AMFR's transmembrane nature limits accessible epitopes

  • Potential cross-reactivity with similar domains in other RING finger proteins

  • Research has shown that even highly selective antibodies like HPA029018 may recognize epitope motifs (e.g., QHA) present in other proteins (NUTM1, RFX6, BRD4)

• Isoform Specificity:

  • Multiple AMFR isoforms with high sequence similarity complicate isoform-specific targeting

  • Differential expression of isoforms across tissues may confound validation

  • Need for careful design to distinguish isoform 1 from isoform 2

• Post-translational Modifications:

  • Glycosylation and other modifications may mask epitopes

  • Modified forms may not be represented in immunization antigens

  • Variable modification states across sample types (e.g., plasma versus tissue lysates)

• Validation Stringency Requirements:

  • Necessity for multiple validation methods beyond traditional Western blotting

  • High-density peptide arrays and protein microarrays containing thousands of antigens (15,728 spots from 13,363 antigens) represent the gold standard for specificity confirmation

  • Need to test across diverse sample types, as AMFR detection patterns differ between plasma and tissue samples

• Reproducibility Across Applications:

  • Antibodies performing well in Western blotting may fail in immunohistochemistry

  • Different fixation methods may affect epitope accessibility

  • Some anti-AMFR antibodies showed reactivity in tissue lysates but not in plasma samples

Addressing these challenges requires comprehensive validation strategies, including the use of AMFR-overexpressing cell lines, knockout controls, and multiple detection technologies.