BECN1 Monoclonal Antibody

What is BECN1 and why is it significant in cellular research?

BECN1 (Beclin 1) is a crucial coiled-coil protein primarily localized in the cytoplasm that serves as the central constituent of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex . This 60 kDa protein plays a fundamental role in autophagosome formation, the key initial step in the autophagy pathway . BECN1's significance extends beyond autophagy regulation to include roles in:

• Maintaining cellular homeostasis through degradation and recycling of damaged proteins and organelles

• Preventing uncontrolled cell growth and tumorigenesis

• Regulating ferroptosis, a form of programmed cell death characterized by lipid peroxidation

• Mediating antiviral host defense mechanisms

• Contributing to developmental processes and neuronal function

BECN1 interacts with numerous protein partners including PIK3C3, ATG14, and UVRAG within the PtdIns3K complex, as well as with regulatory proteins like Bcl-2 that modulate its autophagy-promoting activity . Recent research has revealed BECN1's unexpected interaction with SLC7A11, a component of system Xc-, implicating BECN1 in the regulation of ferroptotic cell death mechanisms .

How do different epitope targets affect BECN1 antibody performance?

The epitope selection significantly impacts experimental outcomes when using BECN1 antibodies. Antibodies targeting the N-terminal region (amino acids 1-150) are particularly valuable for studying BECN1's role in ferroptosis, as this region mediates the interaction with SLC7A11 . In contrast, antibodies recognizing the central coiled-coil domain may be more suitable for investigating canonical autophagy functions. Mouse monoclonal antibodies raised against amino acids 1-300 of human BECN1, such as the G-11 clone, offer versatility across multiple applications including Western blotting, immunoprecipitation, immunofluorescence, and immunohistochemistry .

What are the optimal storage and handling conditions for maintaining BECN1 antibody activity?

BECN1 monoclonal antibodies require specific storage conditions to maintain their binding capacity and specificity. The following protocol is based on experimental optimization with multiple BECN1 antibody clones:

• Storage temperature: Store at -20°C in a non-frost-free freezer to prevent freeze-thaw cycles

• Buffer composition: Phosphate buffered solution (pH 7.4) containing stabilizers (0.05%), protein protectants (0.5%), and glycerol (50%) is optimal for long-term stability

• Aliquoting recommendations: Divide stock solutions into single-use aliquots of 10-20 μL to minimize freeze-thaw cycles

• Working dilution preparation: When preparing working dilutions, use fresh, cold buffer containing 1% BSA or 5% non-fat milk as blocking agents

• Shelf-life considerations: Typical validity period is 12 months when stored according to recommendations

What are the validated protocols for BECN1 detection by Western blotting?

The following protocol has been optimized specifically for BECN1 detection in Western blotting applications:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Include phosphatase inhibitors if studying BECN1 phosphorylation

  • Heat samples at 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol

• Electrophoresis and transfer parameters:

  • Load 20-30 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Transfer to PVDF membrane at 100V for 90 minutes (wet transfer)

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour

  • Incubate with BECN1 primary antibody at 1:1000-2000 dilution overnight at 4°C

  • Wash 3× with TBST, 10 minutes each

  • Incubate with HRP-conjugated secondary antibody at 1:5000 for 1 hour

• Detection considerations:

  • Use enhanced chemiluminescence detection system

  • Expected band size is approximately 60 kDa

  • Multiple bands may indicate post-translational modifications or isoforms

This protocol has been validated using 293T, C2C12, and rat brain samples as positive controls . When troubleshooting, remember that the observed molecular weight may deviate from the expected size due to post-translational modifications, which is common for BECN1 given its regulation by phosphorylation.

How can I optimize immunoprecipitation experiments using BECN1 antibodies?

Immunoprecipitation (IP) is critical for studying BECN1's protein-protein interactions, particularly for investigating novel binding partners like SLC7A11. The following methodology has been validated in multiple cell lines:

• Lysis optimization:

  • Use NP-40 buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0) with protease inhibitors

  • Include 1 mM PMSF, 1 mM NaF, and 1 mM Na3VO4 to preserve phosphorylation states

  • Clear lysates by centrifugation at 14,000 × g for 15 minutes at 4°C

• Pre-clearing strategy:

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

  • Remove 10% of lysate as input control before adding antibody

• Antibody binding parameters:

  • Use 2-5 μg of BECN1 antibody per 500 μg of total protein

  • Incubate overnight at 4°C with gentle rotation

  • Add 40 μL of pre-washed Protein A/G beads and incubate for an additional 2 hours

• Washing and elution:

  • Wash beads 4× with lysis buffer containing decreasing salt concentrations (150 mM to 50 mM)

  • Elute bound proteins with 2× Laemmli buffer at 95°C for 5 minutes

Research has demonstrated that this methodology successfully detects BECN1-SLC7A11 interactions in HCT116 and CX-1 cells following erastin treatment, with enhanced complex formation in BECN1-overexpressing cells . Cross-linking antibodies to beads with dimethyl pimelimidate can reduce background and antibody contamination in the eluate.

What considerations are important for BECN1 immunofluorescence studies?

Immunofluorescence (IF) provides valuable insights into BECN1's subcellular localization and its dynamic redistribution during autophagy induction or stress conditions. The following protocol ensures optimal results:

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes (critical for accessing intracellular epitopes)

  • For detection of membrane-associated BECN1, reduce Triton X-100 concentration to 0.05%

• Blocking and antibody incubation:

  • Block with 5% normal serum (matched to secondary antibody host) for 1 hour

  • Incubate with BECN1 primary antibody at 1:100-200 dilution overnight at 4°C

  • Wash 3× with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated secondary antibody at 1:500 for 1 hour

• Co-localization considerations:

  • For autophagy studies, co-stain with LC3 (autophagosome marker)

  • For organelle co-localization, consider markers for:

    • Golgi apparatus (GM130)

    • Endoplasmic reticulum (Calnexin)

    • Mitochondria (TOM20)

• Imaging parameters:

  • Use confocal microscopy for precise subcellular localization

  • Set exposure times to avoid saturation

  • Collect z-stacks when assessing co-localization

BECN1 typically shows cytoplasmic localization with enrichment at the trans-Golgi network membrane . During autophagy induction, BECN1 redistributes to form punctate structures that correspond to early autophagosome formation sites. Interaction with ATG14 facilitates this translocation to autophagosomes .

How can BECN1 antibodies be used to investigate the role of BECN1 in ferroptosis?

Recent research has revealed an unexpected role for BECN1 in promoting ferroptosis through direct interaction with the cystine/glutamate antiporter system Xc-. BECN1 antibodies can be utilized to investigate this emerging function through several specialized approaches:

• Detecting BECN1-SLC7A11 interactions:

  • Immunoprecipitation assays using anti-BECN1 or anti-SLC7A11 antibodies followed by immunoblot detection have demonstrated significant interaction between these proteins in HCT116 and CX-1 cells following erastin treatment

  • This interaction is enhanced in BECN1-overexpressing cells, suggesting a dose-dependent effect

• Mapping interaction domains:

  • IP assays with truncated BECN1 fragments have shown that the amino acids 1-150 fragment of BECN1 binds to SLC7A11

  • Functional assays confirmed that this N-terminal region enhances erastin-induced cell death and lipid peroxidation similar to full-length BECN1

• Analyzing BECN1 phosphorylation in ferroptosis:

  • BECN1 phosphorylation at S93/96, mediated by AMPK, promotes BECN1-SLC7A11 complex formation and subsequent ferroptosis

  • Western blotting with phospho-specific antibodies can monitor this critical post-translational modification

• Investigating the downstream effects of BECN1 on ferroptosis markers:

  • BECN1 overexpression enhances GSH depletion and increases lipid peroxidation (measured by MDA production) following system Xc- inhibition

  • Knock-down of BECN1 via shRNA confers resistance to system Xc- inhibitors and restores GSH levels

This research approach has revealed that BECN1 plays a dual role in promoting both autophagy and ferroptosis, with distinct protein interactions and post-translational modifications regulating each function.

What techniques are available for studying BECN1 phosphorylation and its functional consequences?

BECN1 function is highly regulated by phosphorylation events that modulate its interactions and subcellular localization. The following techniques enable detailed investigation of these regulatory mechanisms:

• Phospho-specific Western blotting:

  • Use phospho-specific antibodies targeting key sites (S93/96) to monitor AMPK-mediated phosphorylation

  • Include lambda phosphatase-treated controls to confirm phospho-specificity

  • Quantify phosphorylation relative to total BECN1 levels

• Phospho-mutant expression systems:

  • Generate phospho-defective (S90,93,96A) and phospho-mimetic (S90,93,96D/E) BECN1 mutants

  • Express in cell lines with BECN1 knockdown background

  • BECN1 phosphorylation-defective mutants (S90,93,96A) reverse BECN1-induced lipid peroxidation and ferroptosis

• Kinase inhibition studies:

  • Use pharmacological inhibitors (compound C for AMPK) or siRNA-mediated knockdown

  • Monitor effects on BECN1 phosphorylation and downstream functions

  • Inhibition of AMPK by siRNA or compound C diminishes erastin-induced BECN1 phosphorylation and subsequent ferroptosis

• Mass spectrometry analysis:

  • Immunoprecipitate BECN1 from cells treated with various stimuli

  • Perform LC-MS/MS analysis to identify novel phosphorylation sites

  • Quantify changes in phosphorylation stoichiometry using SILAC or TMT labeling

These approaches have revealed that AMPK-mediated phosphorylation of BECN1 at S93/96 is crucial for promoting ferroptosis through enhanced BECN1-SLC7A11 complex formation, while distinct phosphorylation events regulate autophagy induction.

How can I investigate BECN1's role in different protein complexes and cellular pathways?

BECN1 functions in multiple protein complexes to regulate diverse cellular processes. The following methodological approaches enable characterization of these distinct complexes:

• Differential co-immunoprecipitation:

  • Immunoprecipitate BECN1 under different cellular conditions (basal, starvation, oxidative stress)

  • Analyze co-precipitating proteins by Western blot or mass spectrometry

  • Compare interaction partners to identify condition-specific complexes

• Size exclusion chromatography:

  • Fractionate cell lysates based on complex size

  • Analyze fractions by Western blot to identify distinct BECN1-containing complexes

  • Core components of the PtdIns3K complex (PIK3C3, ATG14, UVRAG) will co-fractionate with BECN1

• Proximity labeling approaches:

  • Express BECN1 fused to BioID or APEX2

  • Activate labeling during specific cellular conditions

  • Purify biotinylated proteins and identify by mass spectrometry

• Domain-specific protein interactions:

  • The N-terminal region (aa 1-150) of BECN1 mediates interaction with SLC7A11

  • Central coiled-coil domains mediate dimerization and interaction with ATG14

  • C-terminal regions interact with PI3K components

This multifaceted approach has revealed that BECN1 participates in distinct protein complexes that regulate autophagy, endocytosis, phagocytosis, and ferroptosis in both autophagy-dependent and -independent manners .

Why might I observe multiple bands or unexpected molecular weights when detecting BECN1 by Western blot?

Multiple bands or unexpected molecular weights are common challenges when detecting BECN1 by Western blot. These variations have biological and technical bases:

• Post-translational modifications:

  • Phosphorylation at multiple sites (S93/96, S234, S295) can cause mobility shifts

  • Ubiquitination increases apparent molecular weight by ~8.5 kDa per ubiquitin moiety

  • Other modifications including acetylation and methylation may affect migration

• Proteolytic processing:

  • Caspase-mediated cleavage during apoptosis generates fragments of ~37 kDa

  • Sample preparation without sufficient protease inhibitors may cause artifactual degradation

• Isoforms and splice variants:

  • Multiple BECN1 isoforms exist with varying molecular weights

  • Tissue-specific expression of splice variants can complicate interpretation

• Technical considerations:

  • The observed molecular weight of BECN1 is typically ~60 kDa, which may differ from theoretical predictions

  • Different gel percentages and running conditions affect migration patterns

  • Incomplete denaturation may result in dimeric or oligomeric forms

To confirm band specificity, use multiple BECN1 antibodies targeting different epitopes and include appropriate controls such as BECN1 knockdown or overexpression samples.

How can I distinguish between autophagy-related and ferroptosis-related functions of BECN1?

Distinguishing between BECN1's dual roles in autophagy and ferroptosis requires careful experimental design and specific assays:

• Protein complex analysis:

  • Autophagy: BECN1 interacts with PIK3C3, ATG14, and UVRAG

  • Ferroptosis: BECN1 forms complexes with SLC7A11

  • Use co-immunoprecipitation to determine which complex predominates under specific conditions

• Domain-specific functions:

  • The N-terminal region (aa 1-150) of BECN1 mediates ferroptosis by binding SLC7A11

  • Expression of this fragment enhances erastin-induced cell death and lipid peroxidation

  • C-terminal domains (aa 243-450) are more important for autophagy functions

• Specific inhibitors and assays:

  • Autophagy: Monitor LC3-II formation, p62 degradation, and autophagic flux with bafilomycin A1

  • Ferroptosis: Measure lipid peroxidation (BODIPY-C11, MDA assay), GSH depletion, and test rescue with ferrostatin-1

  • System Xc- function: Measure glutamate release or cystine uptake

• Genetic approaches:

  • Express domain-specific mutants or fragments

  • Use phosphorylation site mutants (S90,93,96A reverses ferroptosis but may not affect autophagy)

  • BECN1 knockdown affects both pathways, while domain-specific mutations can separate functions

Research has demonstrated that AMPK-mediated phosphorylation specifically promotes the ferroptotic function of BECN1 through enhanced BECN1-SLC7A11 complex formation, providing a mechanistic distinction between its dual roles .

How is BECN1 being studied in cancer research and potential therapeutic applications?

BECN1's dual role in autophagy and ferroptosis positions it as a promising target for cancer research and therapy development:

• Tumor suppressor function:

  • Reduced BECN1 expression is observed in various carcinoma cell lines compared to normal tissues

  • This downregulation correlates with tumor progression in lung squamous cell carcinoma and adenocarcinoma

  • Restoring BECN1 expression may inhibit tumor growth through enhanced autophagy

• Ferroptosis induction strategy:

  • Genetic and pharmacological activation of the BECN1 pathway increases ferroptotic cancer cell death in vitro and in vivo

  • Overexpression of BECN1 in tumor cells sensitizes them to system Xc- inhibitors

  • Administration of BECN1 activator peptide (Tat-beclin 1) promotes ferroptosis in mouse models

• Combinatorial approaches:

  • BECN1-mediated ferroptosis works specifically with system Xc- inhibitors (erastin, sulfasalazine, sorafenib)

  • These combinations show efficacy in subcutaneous and orthotopic tumor mouse models

  • The approach specifically increases ferroptotic cell death without affecting apoptosis or necroptosis

• Biomarker potential:

  • BECN1 expression levels may predict responsiveness to ferroptosis-inducing therapies

  • BECN1 phosphorylation status could serve as a biomarker for AMPK activity and potential treatment response

This research suggests that BECN1 activation, particularly in combination with system Xc- inhibitors, represents a promising therapeutic strategy that exploits cancer cells' vulnerability to ferroptotic cell death pathways .

What are the latest techniques for studying BECN1 in neurodegenerative diseases?

BECN1 plays crucial roles in neuronal homeostasis, and its dysfunction is implicated in various neurodegenerative conditions. Advanced techniques for studying BECN1 in neurodegeneration include:

• Neuron-specific expression models:

  • Conditional BECN1 knockout or transgenic models using neuron-specific promoters

  • iPSC-derived neurons from patients with neurodegenerative diseases

  • BECN1 is expressed in dendrites and cell bodies of cerebellar Purkinje cells

• High-resolution imaging approaches:

  • Super-resolution microscopy to visualize BECN1-positive structures in neuronal compartments

  • Live-cell imaging with fluorescently-tagged BECN1 to monitor dynamics during stress

  • Correlative light and electron microscopy (CLEM) to associate BECN1 localization with ultrastructural features

• Disease-specific protein interactions:

  • BECN1 interactions with disease-associated proteins (Tau, α-synuclein, huntingtin)

  • Proximity ligation assays to confirm interactions in brain tissue

  • Mass spectrometry analysis of BECN1 interactome in healthy vs. diseased brain samples

• Tissue-specific analysis:

  • Brain region-specific immunohistochemistry in human brain tissue

  • Laser capture microdissection combined with protein analysis

  • Single-cell approaches to identify cell type-specific BECN1 dysregulation

These approaches have revealed that BECN1 dysfunction contributes to neurodegenerative pathogenesis through impaired clearance of protein aggregates and damaged organelles. Therapeutic strategies targeting BECN1 activation may enhance autophagic clearance mechanisms in neurons, potentially slowing disease progression.

How do BECN1 antibodies contribute to the study of autophagy-independent functions?

Beyond its canonical role in autophagy, BECN1 participates in several autophagy-independent processes that can be investigated using specialized approaches with BECN1 antibodies:

• Endocytosis and receptor trafficking:

  • Use BECN1 antibodies in combination with endocytic markers (EEA1, Rab proteins)

  • Monitor receptor internalization and recycling in BECN1-depleted or overexpressing cells

  • Distinguish from autophagy using ATG5/ATG7 knockout controls

• Antiviral defense mechanisms:

  • BECN1 protects against infection by neurovirulent strains of Sindbis virus

  • Investigate BECN1 interactions with viral proteins using co-immunoprecipitation

  • Monitor viral replication in cells expressing autophagy-defective BECN1 mutants

• Ferroptosis regulation:

  • The BECN1-SLC7A11 interaction regulates ferroptosis independently of canonical autophagy

  • This function is mediated by the N-terminal domain (aa 1-150) of BECN1

  • Use domain-specific antibodies to distinguish autophagy-dependent and -independent functions

• Cell cycle regulation:

  • BECN1 expression is modulated during the cell cycle

  • Use synchronized cell populations and BECN1 antibodies to track levels and modifications

  • Investigate interactions with cell cycle regulatory proteins

These research approaches have revealed BECN1's pleiotropic functions beyond autophagy regulation, highlighting its role as a multifunctional signaling node that integrates various cellular stress responses through distinct protein-protein interactions .

What controls are essential when using BECN1 antibodies for experimental validation?

Proper controls are critical for ensuring the reliability and interpretability of experiments using BECN1 antibodies:

• Antibody specificity controls:

  • BECN1 knockout or knockdown samples (shRNA-mediated as used in experimental validation)

  • Overexpression controls with tagged BECN1 constructs

  • Blocking peptide competition assays to confirm epitope specificity

  • Secondary antibody-only controls to assess non-specific binding

• Experimental treatment controls:

  • For ferroptosis studies: Ferrostatin-1 or iron chelators to confirm ferroptotic mechanism

  • For autophagy studies: Bafilomycin A1 to assess autophagic flux

  • For AMPK-mediated phosphorylation: Compound C as a negative control

• Cell line validation:

  • Multiple cell lines should be tested (HCT116, CX-1, HT1080, PANC1, and Calu-1 have been validated)

  • Primary cells versus immortalized lines may show different BECN1 regulation

  • Tissue-specific expression patterns should be considered

• Biological relevance controls:

  • In vivo validation following in vitro findings

  • Multiple experimental approaches to confirm a finding (e.g., genetic and pharmacological)

  • Time-course experiments to capture dynamic responses

These controls have been successfully implemented in studies demonstrating BECN1's role in ferroptosis, where shRNA-mediated knockdown conferred resistance to system Xc- inhibitors while BECN1 overexpression sensitized cells to these compounds .

How should I design experiments to investigate BECN1 in both in vitro and in vivo settings?

Translating BECN1 research from cell culture to animal models requires careful experimental design:

• In vitro experimental design:

  • Use multiple cell lines relevant to the disease or process under study

  • Employ both genetic (overexpression, knockdown) and pharmacological (Tat-beclin 1) approaches

  • Include time-course and dose-response analyses to capture the dynamics of BECN1-mediated processes

• In vivo model selection:

  • Subcutaneous tumor models provide accessibility for monitoring growth and drug delivery

  • Orthotopic models better recapitulate the native tumor microenvironment

  • Genetically engineered mouse models with tissue-specific BECN1 modulation offer physiological relevance

• Translational considerations:

  • BECN1 activator peptides (Tat-beclin 1) have shown efficacy in promoting ferroptosis in vivo

  • Combination approaches with system Xc- inhibitors enhance therapeutic effects

  • Monitor both target engagement (BECN1 phosphorylation, complex formation) and phenotypic outcomes

• Analytical approaches:

  • Immunohistochemistry with validated antibodies for tissue analysis

  • Biochemical assays from tissue lysates (co-immunoprecipitation, Western blotting)

  • Multi-parameter analysis (tumor growth, lipid peroxidation, GSH levels, autophagic markers)