AMBRA1 Antibody

What is AMBRA1 and why is it significant in research?

AMBRA1 (Activating molecule in BECN1-regulated autophagy protein 1), also known as WDR94 or KIAA1736, is a 1,298 amino acid protein characterized by three WD repeats that form a tertiary propeller structure. This structure is crucial for AMBRA1's diverse cellular functions, including autophagy regulation, cell proliferation, and survival during neuronal development . AMBRA1 primarily localizes to cytoplasmic vesicles, where it plays a significant role in controlling protein turnover and facilitating nervous system development . Its importance in autophagy regulation makes it a potential therapeutic target for neurodegenerative diseases where autophagy dysregulation is often observed .

What applications are AMBRA1 antibodies suitable for?

AMBRA1 antibodies have been validated for multiple experimental applications:

The antibody can detect endogenous levels of AMBRA1 protein in human, mouse, and rat samples .

How should AMBRA1 antibodies be stored for optimal performance?

For short-term storage, AMBRA1 antibodies should be stored at 4°C. For long-term storage, it is recommended to aliquot the antibody and store at -20°C to avoid freeze-thaw cycles, which can compromise antibody performance . The formulation typically includes PBS with 30% glycerol and 0.05% sodium azide as a preservative . Always check manufacturer-specific recommendations as formulations may vary.

What are the key considerations for successful Western blotting with AMBRA1 antibodies?

When performing Western blotting for AMBRA1:

• Expected molecular weight is between 135-150 kDa

• Sample preparation is critical: use fresh tissue/cell lysates with protease inhibitors

• Include positive controls (e.g., mouse liver lysate has been validated for AMBRA1 detection)

• Use appropriate blocking agents to minimize background

• Secondary antibody selection should match the host species of the primary antibody (typically rabbit or mouse for commercial AMBRA1 antibodies)

• Extended transfer times may be necessary due to the high molecular weight of AMBRA1

How can researchers distinguish between AMBRA1 isoforms in experimental settings?

Multiple AMBRA1 isoforms arise from alternative splicing events, potentially contributing to diverse functional roles in cellular homeostasis and stress responses . To distinguish between isoforms:

• Use high-resolution SDS-PAGE with extended run times to separate closely migrating bands

• Consider using isoform-specific antibodies if available (check epitope information)

• Employ 2D gel electrophoresis to separate isoforms based on both molecular weight and isoelectric point

• Validate findings with recombinant expression of specific isoforms as controls

• For definitive identification, consider mass spectrometry analysis of immunoprecipitated protein

For nuclear vs. cytoplasmic isoforms, subcellular fractionation followed by Western blotting has been successfully used to demonstrate that AMBRA1 is present at comparable levels in cytosolic and nuclear fractions, with even higher levels in the perinuclear fraction .

What approaches can be used to study AMBRA1's dual role in autophagy and gene expression?

AMBRA1 has been reported to function both in autophagy regulation and gene expression. To study this dual functionality:

• For autophagy studies:

  • Monitor AMBRA1 interaction with BECN1-PIK3C3 complex through co-immunoprecipitation

  • Assess autophagy flux using LC3 conversion assays in the presence/absence of AMBRA1

  • Utilize fluorescently-tagged AMBRA1 constructs to track localization during autophagy induction

• For gene expression studies:

  • Perform chromatin immunoprecipitation (ChIP) to identify AMBRA1-associated genomic regions

  • Use nuclear extraction protocols followed by immunoprecipitation and mass spectrometry to identify nuclear binding partners, as was done in squamous cell carcinoma cells

  • Employ RNA-seq following AMBRA1 knockdown/overexpression to identify regulated genes

• For integrated studies:

  • Create domain-specific mutants to dissect which regions are responsible for each function

  • Use proximity labeling techniques (BioID or APEX) to identify compartment-specific interaction partners

Research has shown that AMBRA1 is detected in nuclear extracts and interacts with proteins involved in transcription, suggesting a direct role in gene regulation beyond its cytoplasmic autophagy functions .

What are the critical controls needed when using AMBRA1 antibodies for subcellular localization studies?

When performing subcellular localization studies with AMBRA1 antibodies:

• Essential controls:

  • Negative controls: secondary antibody only, isotype control, and AMBRA1 knockdown samples

  • Subcellular fraction purity controls: blot for compartment-specific markers such as:

    • GM130 (Golgi apparatus)

    • PDI (endoplasmic reticulum)

    • Lamin A/C (nuclear envelope)

    • GAPDH (cytosol)

  • Peptide competition assay to confirm antibody specificity

  • Multiple antibodies targeting different AMBRA1 epitopes to confirm localization pattern

• Validation approaches:

  • Correlate antibody staining with tagged AMBRA1 overexpression

  • Compare results across multiple cell types (e.g., SCC cell lines and primary keratinocytes show different nuclear AMBRA1 patterns)

  • Validate findings with orthogonal techniques (fractionation + Western blot vs. immunofluorescence)

Fraction purity confirmation is particularly important as demonstrated in studies showing AMBRA1 in both cytoplasmic and nuclear compartments .

What are common troubleshooting strategies for weak or non-specific AMBRA1 antibody signals?

When facing challenges with AMBRA1 detection:

• For weak signals:

  • Optimize antibody concentration (try a titration between 1:500-1:2000 for Western blot)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider using signal enhancement systems (HRP amplification)

  • For IHC/ICC applications, ensure proper antigen retrieval (sodium citrate buffer pH 6.0 is recommended)

  • Check protein extraction method (AMBRA1 is associated with membrane structures and may require detergent-based extraction)

• For non-specific signals:

  • Increase washing steps duration and frequency

  • Optimize blocking conditions (try 5% BSA instead of milk for phosphorylated epitopes)

  • Use more stringent washing buffers (increase Tween-20 concentration)

  • Consider using monoclonal antibodies like AMBRA1 Antibody (G-6) for higher specificity

  • Pre-absorb antibody with recombinant protein if available

• For inconsistent results:

  • Test antibody performance in multiple validated positive control samples

  • Consider lot-to-lot variations (request same lot for critical experiments)

  • Ensure proper storage conditions to maintain antibody integrity

How should researchers address potential cross-reactivity between AMBRA1 and other WD40-domain containing proteins?

Since AMBRA1 contains WD40 domains that are found in many proteins, cross-reactivity is a legitimate concern:

• Validation approaches:

  • Perform parallel experiments with AMBRA1 knockdown/knockout samples

  • Compare results from antibodies targeting different AMBRA1 epitopes

  • Conduct immunoprecipitation followed by mass spectrometry to confirm target identity

  • Look for the distinctive molecular weight pattern (~142 kDa for full-length AMBRA1)

• Specificity enhancement:

  • Use affinity-purified antibodies when available

  • Consider monoclonal antibodies for critical applications

  • Perform pre-absorption with recombinant WD40-domain proteins to remove cross-reactive antibodies

  • Analyze antibody epitope sequences for uniqueness within the WD40 protein family

• Confirmatory strategies:

  • Correlate antibody detection with orthogonal methods (qPCR for mRNA expression)

  • Perform reciprocal detection with multiple antibodies recognizing different epitopes

  • Use recombinant expression systems with tagged AMBRA1 as positive controls

How can AMBRA1 antibodies be utilized to study its role in cancer progression?

Recent research indicates AMBRA1 functions as a tumor suppressor that promotes genomic integrity during DNA replication . To investigate its role in cancer:

• Tumor expression profiling:

  • Use IHC with AMBRA1 antibodies to compare expression in tumor vs. normal tissue

  • Correlate expression levels with patient outcomes in tissue microarrays

  • Examine subcellular localization changes in different cancer stages

• Functional studies:

  • Combine AMBRA1 antibody-based detection with markers of autophagy, cell cycle, and apoptosis

  • Use proximity ligation assays to detect AMBRA1 interactions with cancer-related proteins

  • Study AMBRA1's interaction with DCX E3 ubiquitin-protein ligase complexes that regulate cell cycle control

• Mechanistic investigations:

  • Immunoprecipitate AMBRA1 to identify cancer-specific binding partners

  • Study AMBRA1's role in regulating cyclin-D (CCND1, CCND2, CCND3) degradation

  • Investigate how AMBRA1 promotes MYC dephosphorylation and degradation

Research has shown AMBRA1 drives gastric cancer progression through tumor plasticity regulation , making it an important target for oncology research.

What experimental approaches can be used to study AMBRA1's role in autophagy regulation?

To investigate AMBRA1's function in autophagy:

• Protein interaction studies:

  • Co-immunoprecipitation of AMBRA1 with BECN1-PIK3C3 complex components

  • Use proximity labeling techniques to identify transient interactors during autophagy induction

  • Map interaction domains through truncation mutants and co-IP

• Functional autophagy assays:

  • Monitor LC3-I to LC3-II conversion in AMBRA1-depleted cells

  • Track autophagy flux using tandem fluorescent-tagged LC3 reporters with/without AMBRA1

  • Quantify autophagic vesicles using fluorescence microscopy after AMBRA1 modulation

• Regulatory mechanism studies:

  • Investigate AMBRA1 phosphorylation status using phospho-specific antibodies

  • Examine AMBRA1's regulation of ULK1 ubiquitination

  • Study AMBRA1's role in selective autophagy pathways (mitophagy, ER-phagy)

• Therapeutic implications:

  • Screen for compounds that modulate AMBRA1-dependent autophagy

  • Test autophagy modulators in neurodegenerative disease models where AMBRA1 functions

How can researchers investigate the nuclear functions of AMBRA1?

Recent studies have revealed AMBRA1 also localizes to the nucleus and may regulate gene expression . To explore this function:

• Nuclear localization studies:

  • Perform subcellular fractionation followed by Western blotting

  • Use immunofluorescence with confocal microscopy for co-localization with nuclear markers

  • Identify nuclear localization signals within AMBRA1 using mutational analysis

• Chromatin association:

  • Perform chromatin immunoprecipitation (ChIP) to identify AMBRA1-associated genomic regions

  • Use ChIP-seq to map genome-wide binding patterns

  • Conduct DNA pull-down assays to test direct DNA binding capability

• Nuclear protein interactions:

  • Conduct nuclear co-immunoprecipitation followed by mass spectrometry

  • Previous studies identified 456 Ambra1-interacting proteins in the nucleus, including nuclear pore complex components

  • Use proximity-dependent biotin identification (BioID) with nuclear-targeted AMBRA1

• Transcriptional impact assessment:

  • Perform RNA-seq after nuclear AMBRA1 depletion

  • Use reporter assays to test AMBRA1's effect on specific promoters

  • Investigate changes in chromatin accessibility using ATAC-seq

How can AMBRA1 antibodies be applied to study neurodevelopmental processes?

AMBRA1 is expressed during neurodevelopment and is required for neural tube development . Researchers can:

• Developmental profiling:

  • Use IHC with AMBRA1 antibodies to map expression patterns during brain development

  • Perform co-localization studies with neural stem cell and differentiation markers

  • Quantify AMBRA1 levels during critical developmental windows

• Functional studies in neural models:

  • Apply AMBRA1 antibodies in neuronal primary cultures to track subcellular distribution

  • Use ICC/IF in iPSC-derived neurons to study AMBRA1 localization during differentiation

  • Combine with autophagy assays to link developmental phenotypes with autophagic function

• Pathological investigations:

  • Compare AMBRA1 expression in neurodevelopmental disorders

  • Study interaction with disease-associated proteins

  • Assess post-translational modifications in pathological conditions

The double labeling technique using AMBRA1 antibody (green) and PV (red) in CA1 neurons with DAPI counterstain has been successfully applied to visualize AMBRA1 expression in specific neuronal populations .

What considerations are important when using AMBRA1 antibodies for multi-omics integration studies?

For researchers combining antibody-based detection with other omics approaches:

• Integration with proteomics:

  • Use AMBRA1 immunoprecipitation followed by mass spectrometry for interactome analysis

  • Consider antibody-based enrichment prior to proteomic analysis

  • Validate mass spectrometry findings with targeted antibody detection

• Correlation with transcriptomics:

  • Compare protein-level changes detected by AMBRA1 antibodies with mRNA expression data

  • Account for potential post-transcriptional regulation when interpreting discrepancies

  • Use subcellular localization data to refine functional predictions from expression data

• Connection to functional genomics:

  • Combine CRISPR screens with AMBRA1 antibody-based phenotypic readouts

  • Link genetic variants to protein expression or localization changes

  • Correlate epigenetic modifications with AMBRA1 expression patterns

• Quality control considerations:

  • Document antibody validation data thoroughly for integration with other omics datasets

  • Consider batch effects when comparing antibody-based data across experiments

  • Establish standardized protocols for sample processing across platforms