Phospho-BCL2L1 (S62) Antibody

What is the specificity of Phospho-BCL2L1 (S62) Antibody?

Phospho-BCL2L1 (S62) antibody specifically recognizes BCL2L1 (also known as Bcl-xL, Bcl-2-like protein 1, or apoptosis regulator Bcl-X) only when phosphorylated at serine 62. This phospho-specific antibody is generated using synthetic phosphopeptides corresponding to residues surrounding Ser62 of human Bcl-xL as the immunogen . The specificity is important for distinguishing the phosphorylated form from unphosphorylated BCL2L1, allowing researchers to track the phosphorylation state of this protein under various experimental conditions. Unlike general BCL2L1 antibodies, this phospho-specific variant enables precise detection of the post-translational modification that governs specific protein functions .

What are the optimal experimental applications for Phospho-BCL2L1 (S62) Antibody?

Phospho-BCL2L1 (S62) antibody has been validated for multiple experimental applications with specific recommended dilutions:

These applications allow researchers to detect and quantify phosphorylated BCL2L1 in various experimental contexts, from protein extracts (WB) to visualizing its subcellular localization in fixed cells (IF/ICC) or tissues (IHC) . Western blotting is particularly useful for observing the 30 kDa molecular weight band corresponding to phosphorylated BCL2L1 .

What species reactivity can be expected with Phospho-BCL2L1 (S62) Antibody?

The commercially available Phospho-BCL2L1 (S62) antibodies consistently show reactivity with human, mouse, and rat samples . This cross-species reactivity is due to the high conservation of the amino acid sequence surrounding the Ser62 phosphorylation site across these mammalian species. The conservation allows researchers to use the same antibody for experiments with various model systems, facilitating comparative studies between human samples and common laboratory animal models . For other species, validation experiments should be performed before assuming cross-reactivity.

What are the proper storage conditions for Phospho-BCL2L1 (S62) Antibody?

For optimal performance and longevity of Phospho-BCL2L1 (S62) antibody, proper storage is essential:

• Store at -20°C upon receipt

• Some sources recommend -80°C for long-term storage

• Avoid repeated freeze/thaw cycles that can degrade antibody activity

• The antibody is typically provided in a buffer containing PBS (pH 7.3), 50% glycerol, and 0.02% sodium azide, which helps maintain stability

• Some formulations may also contain BSA (0.5%) as a stabilizer

• When working with the antibody, it's advisable to make small aliquots to minimize freeze/thaw cycles

Following these storage guidelines will help maintain antibody performance and extend its useful shelf life for research applications .

How does phosphorylation at Ser62 alter BCL2L1 function during mitosis?

Phosphorylation of BCL2L1 at Ser62 plays a critical role in regulating mitotic progression through distinct mechanisms:

During mitosis, BCL2L1(S62) is strongly phosphorylated by PLK1 (Polo-like kinase 1) and MAPK14/stress-activated protein kinase p38α (SAPKp38α) specifically during prophase, prometaphase, and at the metaphase/anaphase boundaries . This phosphorylation exhibits a dynamic pattern, with rapid dephosphorylation occurring at telophase and cytokinesis .

The phosphorylated form of BCL2L1(S62) exhibits distinct subcellular localization during mitosis, concentrating in centrosomes with γ-tubulin and in mitotic cytosol with spindle-assembly checkpoint (SAC) signaling components, including PLK1, BubR1, and Mad2 . This suggests a non-apoptotic role in mitotic regulation.

In taxol- and nocodazole-treated cells, phospho-BCL2L1(S62) associates with Cdc20-, Mad2-, BubR1-, and Bub3-complexes, while the non-phosphorylatable mutant BCL2L1(S62A) fails to bind these complexes . This indicates that phosphorylation at this site is necessary for these protein-protein interactions.

Functionally, cells expressing wild-type BCL2L1 (which can be phosphorylated at S62) undergo normal mitosis, while expression of the non-phosphorylatable S62A mutant leads to multiple mitotic defects, including delayed anaphase and chromosome mis-segregation . This demonstrates that the phosphorylation state at this specific residue is crucial for proper mitotic progression.

What methods can be used to validate the specificity of Phospho-BCL2L1 (S62) Antibody in experimental systems?

Validating antibody specificity is crucial for reliable experimental results. For Phospho-BCL2L1 (S62) antibody, several approaches can be employed:

• Phosphatase treatment control: Treating cell lysates with lambda phosphatase before Western blotting should eliminate the signal from a truly phospho-specific antibody.

• Phosphorylation site mutants: Expressing BCL2L1 with S62A mutation (non-phosphorylatable) or S62D mutation (phosphomimetic) can serve as negative and positive controls, respectively. The antibody should not recognize the S62A mutant but may recognize the S62D mutant .

• Cell cycle synchronization: Since BCL2L1(S62) phosphorylation increases during specific phases of mitosis, comparing lysates from cells synchronized at different cell cycle stages can validate the antibody's ability to detect these dynamic changes .

• Kinase inhibition: Treatment with PLK1 or p38 inhibitors should reduce the signal detected by the antibody, as these are the kinases responsible for S62 phosphorylation .

• Blocking peptide competition: Pre-incubating the antibody with the phospho-peptide used as the immunogen should block specific binding in subsequent applications.

• Cross-reactivity testing: Testing against related phosphorylated BCL2 family members can confirm specificity for BCL2L1 over other family proteins.

The combination of these approaches provides strong validation of antibody specificity, essential for publishing reliable research results .

How does BCL2L1 phosphorylation at Ser62 differ functionally from other phosphorylation sites in the protein?

BCL2L1 undergoes phosphorylation at multiple sites, each with distinct functional consequences:

Ser62 phosphorylation is primarily associated with mitotic regulation. As described previously, it regulates interactions with spindle assembly checkpoint proteins and is critical for proper mitotic progression . This phosphorylation is mediated by PLK1 and p38 kinases and follows a specific temporal pattern during mitosis .

In contrast, Ser49 phosphorylation follows a different temporal pattern during the cell cycle. Phospho-BCL2L1(S49) appears during S and G2 phases but disappears during early mitosis (prophase, prometaphase, and metaphase) before reappearing during anaphase, telophase, and cytokinesis . PLK3 is the key kinase responsible for S49 phosphorylation, distinguishing it mechanistically from S62 phosphorylation .

Thr47 and Thr115 represent additional phosphorylation sites on BCL2L1 with their own regulatory functions . These sites may influence protein stability, interaction partners, or anti-apoptotic activity through distinct mechanisms.

Understanding the interplay between these different phosphorylation events is critical for dissecting the complex regulation of BCL2L1 function. For complete functional characterization, researchers should consider examining multiple phosphorylation sites simultaneously and their potential cooperative or antagonistic effects .

What is the relationship between BCL2L1(S62) phosphorylation and the regulation of mitophagy?

Research has revealed a surprising connection between BCL2L1 phosphorylation and the regulation of mitophagy (selective autophagy of mitochondria):

BCL2L1, but not BCL2, strongly suppresses FUNDC1-mediated mitophagy, a critical mechanism for removing damaged mitochondria in response to cellular stress . This function appears to be independent of BCL2L1's well-known anti-apoptotic activity.

Mechanistically, BCL2L1 inhibits PGAM5, a mitochondrially localized phosphatase that dephosphorylates FUNDC1 at Ser13 . Since dephosphorylated FUNDC1 activates hypoxia-induced mitophagy, BCL2L1's inhibition of PGAM5 prevents this activation.

Importantly, this function of BCL2L1 in regulating mitophagy through the PGAM5-FUNDC1 axis is distinct from its role in apoptosis regulation . The BCL2L1-PGAM5-FUNDC1 signaling pathway represents a novel mechanism of mitochondrial quality control.

Under hypoxic conditions, knockdown of BCL2L1 enhances mitophagy, while overexpression of BCL2L1 inhibits it . This suggests that modulating BCL2L1 levels or its phosphorylation state could be a potential therapeutic approach for conditions associated with mitochondrial dysfunction.

The exact role of S62 phosphorylation in this context requires further investigation, but these findings highlight the multifunctional nature of BCL2L1 beyond its classical role in apoptosis regulation .

What technical considerations are important when performing co-immunoprecipitation with Phospho-BCL2L1 (S62) Antibody?

When designing co-immunoprecipitation (co-IP) experiments with Phospho-BCL2L1 (S62) antibody, several technical aspects require attention:

• Buffer composition: For BCL2L1 co-IP, two different buffer systems have been successfully used: MSH buffer (210 mM mannitol, 70 mM sucrose, 20 mM HEPES, 1 mM EDTA, pH 7.4) with 1% CHAPS or 1% Nonidet P-40 buffer . The choice may depend on the specific interaction partners being investigated.

• Phosphatase inhibitors: Including phosphatase inhibitors (such as PhosStop™) in lysis buffers is critical for preserving the phosphorylation state of BCL2L1 . Without these inhibitors, rapid dephosphorylation can occur during sample preparation.

• Antibody coupling: For optimal results, 2 μg of rabbit anti-BCL2L1 antibody can be covalently coupled to Tachisorb™-immunoadsorbent or similar matrix . This coupling can be performed using dimethylpimelidate dihydrochloride in borax buffer (pH 9.0) .

• Incubation conditions: For pull-down of interacting proteins, coupled antibodies should be added to lysates for 6 hours with rotation at 4°C . This extended incubation helps capture transient or weak interactions.

• Expected interaction partners: Depending on the experimental context, co-IP with phospho-BCL2L1(S62) antibody may pull down various interacting proteins including components of the spindle assembly checkpoint machinery (Mad2, BubR1, Bub3) in mitotic cells , or proteins involved in mitophagy regulation such as PGAM5 .

• Control immunoprecipitations: Using rabbit immunoglobulin as a negative control is essential to identify non-specific binding . Additionally, comparing wild-type BCL2L1 to S62A mutant can help confirm phosphorylation-dependent interactions.

These methodological considerations will help ensure successful co-IP experiments for investigating phosphorylation-dependent protein interactions of BCL2L1 .

How can researchers overcome weak or absent signals when using Phospho-BCL2L1 (S62) Antibody in Western blots?

When facing weak or absent signals in Western blots using Phospho-BCL2L1 (S62) antibody, consider these optimization strategies:

• Sample preparation: Include phosphatase inhibitors (e.g., PhosStop™) in lysis buffers to prevent dephosphorylation during sample preparation . Process samples quickly and keep them cold throughout preparation.

• Antibody dilution optimization: While recommended dilutions range from 1:500 to 1:2000 for Western blotting , testing a more concentrated dilution (e.g., 1:250) may help detect weak signals.

• Protein loading: Increase the amount of total protein loaded (e.g., 50-100 μg instead of 20-30 μg) to enhance detection of low-abundance phosphorylated species.

• Enhanced detection systems: Use high-sensitivity chemiluminescence substrates like SuperSignal WestPico or ECL Plus rather than standard ECL reagents.

• Enrichment strategies: Consider phosphoprotein enrichment methods or immunoprecipitation prior to Western blotting to concentrate the phosphorylated target.

• Biological induction: Treat cells with conditions known to increase S62 phosphorylation, such as microtubule-disrupting agents (taxol, nocodazole) that arrest cells in mitosis when this phosphorylation is highest .

• Positive controls: Include lysates from cells with known high levels of BCL2L1(S62) phosphorylation (e.g., mitotically arrested cells) as positive controls .

• Membrane type: PVDF membranes may provide better results than nitrocellulose for detecting some phosphoproteins.

• Blocking optimization: Consider using 5% BSA instead of milk for blocking, as milk contains phosphoproteins that may interfere with phospho-specific antibody binding.

• Extended exposure times: For digital imaging systems, try longer exposure times to capture weak signals.

Implementing these strategies should help troubleshoot and optimize Western blot detection of phosphorylated BCL2L1(S62) .

What controls should be included when studying the effects of BCL2L1(S62) phosphorylation on cellular processes?

To rigorously investigate the effects of BCL2L1(S62) phosphorylation on cellular processes, incorporating these controls is essential:

• Phosphorylation site mutants: Include both S62A (non-phosphorylatable) and S62D (phosphomimetic) mutants alongside wild-type BCL2L1 . These allow for distinguishing effects that are specifically dependent on the phosphorylation state at S62.

• BCL2 expression control: Since BCL2L1 (but not BCL2) has been shown to regulate processes like mitophagy , including BCL2 expression controls can help distinguish BCL2L1-specific functions from general anti-apoptotic effects.

• Expression level controls: Ensure that wild-type and mutant proteins are expressed at comparable levels. Western blotting with total BCL2L1 antibodies can confirm that observed phenotypic differences are not due to different expression levels .

• Kinase inhibition controls: Include conditions where the kinases responsible for S62 phosphorylation (PLK1, p38) are inhibited pharmacologically to corroborate phenotypes observed with non-phosphorylatable mutants .

• Cell cycle synchronization: When studying mitotic effects, proper synchronization controls are critical, as BCL2L1 phosphorylation varies throughout the cell cycle. Include markers like phospho-histone H3 to confirm mitotic status .

• Functional readouts: Depending on the process being studied, include appropriate readouts such as:

  • For mitotic effects: chromosome segregation analysis, mitotic index, spindle assembly checkpoint function

  • For apoptosis: caspase activation, PARP cleavage, annexin V staining

  • For mitophagy: mitochondrial content, LC3 puncta formation, degradation of mitochondrial proteins

• Rescue experiments: In knockdown/knockout experiments, rescue with wild-type versus S62A or S62D mutants can definitively demonstrate phosphorylation-dependent functions .

These controls provide a comprehensive framework for establishing causal relationships between BCL2L1(S62) phosphorylation and specific cellular phenotypes .

How can researchers distinguish between the effects of BCL2L1(S62) phosphorylation on apoptosis versus its non-apoptotic functions?

Distinguishing between apoptotic and non-apoptotic functions of BCL2L1(S62) phosphorylation requires careful experimental design:

• Time-course analyses: BCL2L1's role in mitotic regulation occurs during cell division, while its anti-apoptotic functions may be more relevant during stress. Temporal analysis can help separate these functions .

• Cell death-independent assays: For studying non-apoptotic functions like mitotic regulation or mitophagy, employ assays that don't rely on cell viability as an endpoint:

  • For mitotic functions: time-lapse imaging of chromosome segregation, spindle assembly checkpoint activity measurements

  • For mitophagy: monitor mitochondrial content, LC3-II conversion, or mitochondrial protein degradation independent of cell death

• BH3 domain mutants: The research shows that the BH3 domain of BCL2L1 is important for its inhibitory action on mitophagy, yet BH3 domain deletion mutants maintained their anti-apoptotic role . Using such constructs can help separate these functions.

• BECN1 dependency tests: BCL2L1 inhibition of general autophagy is BECN1-dependent, while its inhibition of mitophagy appears BECN1-independent . Testing in BECN1 knockdown cells can distinguish between these pathways.

• Combined apoptosis and function assays: Simultaneously assess apoptotic markers (annexin V, PARP cleavage) alongside functional endpoints (mitotic progression, mitophagy) in the same experimental system .

• Context-specific experiments: Study BCL2L1(S62) phosphorylation in contexts where apoptosis is not actively engaged, such as in normal mitotic progression in the absence of cellular stress .

• Caspase inhibition: Use broad-spectrum caspase inhibitors (e.g., z-VAD-fmk) to block apoptosis execution while monitoring non-apoptotic functions. This can help separate downstream effects of cell death from direct effects of BCL2L1 phosphorylation .

Research demonstrates that under hypoxic conditions, BCL2L1 knockdown enhances mitophagy without affecting apoptosis, and this can be assessed by monitoring phosphatidylserine exposure and cytochrome c release . These approaches allow for dissection of the multiple functions of BCL2L1(S62) phosphorylation beyond its classical role in apoptosis regulation .

How can Phospho-BCL2L1 (S62) Antibody be used to investigate the role of BCL2L1 in cancer biology?

Phospho-BCL2L1 (S62) antibody offers valuable applications for cancer research:

• Prognostic biomarker exploration: The phosphorylation status of BCL2L1 at S62 may correlate with cancer progression, therapy resistance, or patient outcomes. Immunohistochemistry in tissue microarrays can assess this potential biomarker in patient samples .

• Drug response monitoring: Since mitotic arrest induces complete phosphorylation of BCL2L1 at S62 by cyclin-dependent kinase 1 (CDK1), inactivating its anti-apoptotic function , monitoring this phosphorylation can help assess the cellular response to anti-mitotic drugs like taxanes.

• Combination therapy rationale: Understanding how BCL2L1(S62) phosphorylation affects both apoptosis sensitivity and mitotic progression can inform rational combination therapies targeting both processes simultaneously .

• Mitotic checkpoint dysregulation: Since phospho-BCL2L1(S62) interacts with spindle assembly checkpoint components, investigating these interactions in cancer cells with chromosomal instability could reveal novel therapeutic vulnerabilities .

• Mitophagy regulation in cancer: The role of BCL2L1 in suppressing mitophagy via the PGAM5-FUNDC1 axis suggests that cancer cells might exploit this pathway to maintain mitochondrial function despite adverse conditions like hypoxia.

• Cell line models: In neuroblastoma cell lines like SH-EP, LAN-1, SKNSH, SH-SY5Y, IMR-32, STA-NB1, STA-NB3, and STA-NB15, BCL2L1 phosphorylation may influence response to proteasome inhibitors like bortezomib , providing model systems for investigating these mechanisms.

The unique ability of phospho-specific antibodies to track the dynamic post-translational modifications of BCL2L1 enables researchers to explore its complex roles in cancer biology beyond simple expression level analysis .

What is the significance of observed differences between calculated (26 kDa) and observed (30 kDa) molecular weights for BCL2L1?

The discrepancy between the calculated (26 kDa) and observed (30 kDa) molecular weight of BCL2L1 in SDS-PAGE warrants scientific consideration:

• Post-translational modifications: Phosphorylation at S62 and potentially at other sites (S49, T47, T115) contributes to reduced electrophoretic mobility . Multiple phosphorylation events can significantly alter migration patterns.

• Protein structure influence: The highly helical structure of BCL2L1 may cause anomalous migration in SDS-PAGE. Proteins with high α-helical content often migrate more slowly than predicted from their sequence alone.

• Technical validation: Researchers should confirm that the observed 30 kDa band is indeed BCL2L1 through techniques such as:

  • Immunoblotting with multiple BCL2L1 antibodies targeting different epitopes

  • Mass spectrometry identification of the protein band

  • Comparing migration patterns of recombinant protein vs. endogenous protein

  • Examining migration shifts with phosphatase treatment

• Experimental implications: When analyzing Western blots, researchers should look for the 30 kDa band rather than at the calculated 26 kDa position . This knowledge prevents misinterpretation of results or concerns about antibody specificity.

• Isoform consideration: BCL2L1 has multiple isoforms (including BCL-xL and BCL-xS), and the observed molecular weight may reflect the specific isoform present in the experimental system .

This migration discrepancy exemplifies the importance of empirical validation in protein research and highlights how post-translational modifications can significantly impact protein characteristics detectable by biochemical methods .

How can dynamic phosphorylation patterns of BCL2L1 be accurately assessed throughout the cell cycle?

Tracking the dynamic phosphorylation of BCL2L1 throughout the cell cycle requires sophisticated approaches:

• Synchronized cell populations: Use methods like double thymidine block, nocodazole arrest/release, or mitotic shake-off to obtain populations enriched at specific cell cycle stages . Validate synchronization with flow cytometry for DNA content and cell cycle markers.

• Time-resolved sampling: Collect samples at close intervals (e.g., every 15-30 minutes) after synchronization release to capture rapid phosphorylation/dephosphorylation events, particularly during mitotic transitions .

• Multiplexed phospho-antibodies: Simultaneously probe for phospho-BCL2L1(S62) and phospho-BCL2L1(S49) to compare their distinct temporal patterns . Include cell cycle phase markers such as:

  • G2 phase: Cyclin B1

  • Mitosis entry: phospho-histone H3 (Ser10)

  • Mitotic progression: phospho-PLK1, cyclin B1 degradation

  • Mitotic exit: decreased CDK1 activity

• Quantitative immunofluorescence: Perform immunostaining with phospho-specific antibodies in fixed cells, coupled with DNA staining. This allows for single-cell resolution analysis of phosphorylation levels correlated with cell cycle position .

• Live-cell imaging: For real-time tracking, consider developing a phosphorylation biosensor system based on FRET (Förster Resonance Energy Transfer) that can report on BCL2L1 phosphorylation status in living cells.

• Subcellular localization: Track not only the phosphorylation levels but also the subcellular distribution of phosphorylated BCL2L1, which changes through the cell cycle . Co-staining with centrosomal markers (γ-tubulin), mitotic spindle components, or midbody markers provides spatial context.

• Mass spectrometry approach: For comprehensive analysis, use phospho-proteomics with synchronized cell populations to quantitatively track multiple phosphorylation sites simultaneously.

Research has shown that phospho-BCL2L1(S49) appears during S and G2 phases but disappears in early mitosis, while phospho-BCL2L1(S62) is strongly present during prophase, prometaphase, and metaphase/anaphase boundaries . These distinct patterns require careful temporal resolution for accurate characterization .

What potential exists for targeting BCL2L1 phosphorylation as a therapeutic strategy?

Targeting BCL2L1 phosphorylation offers several promising therapeutic avenues:

• Mitotic vulnerability exploitation: Since phosphorylation at S62 by CDK1 inactivates the anti-apoptotic function of BCL2L1 , combining mitotic inhibitors (which induce S62 phosphorylation) with additional apoptotic stimuli could create a synthetic lethal interaction in cancer cells.

• Kinase inhibitor combinations: Targeting the kinases responsible for BCL2L1 phosphorylation (PLK1, p38, CDK1) in combination with BCL2 family inhibitors may provide synergistic therapeutic effects . This approach could simultaneously disrupt both the anti-apoptotic function and the mitotic regulatory role of BCL2L1.

• Mitophagy modulation: Given BCL2L1's role in inhibiting FUNDC1-mediated mitophagy via the PGAM5 phosphatase , therapeutic strategies that either enhance or inhibit this pathway could be beneficial depending on the disease context:

  • In cancer: Enhancing mitophagy might eliminate damaged mitochondria that cancer cells rely on

  • In neurodegenerative diseases: Inhibiting excessive mitophagy might be neuroprotective

• Phosphorylation-state specific targeting: Developing compounds that specifically recognize and bind to phosphorylated BCL2L1(S62) could selectively target cells with high mitotic activity, potentially with fewer side effects than general anti-mitotic drugs.

• BH3 domain interactions: Research shows that the BH3 domain of BCL2L1 is critical for its inhibition of mitophagy but dispensable for its anti-apoptotic function . Compounds targeting this specific domain could selectively modulate mitophagy without affecting apoptosis.

• Cell cycle checkpoint manipulation: Since phospho-BCL2L1(S62) interacts with spindle assembly checkpoint proteins , targeting these interactions could potentially sensitize cancer cells to mitotic catastrophe.

The therapeutic potential is supported by observations that BCL2L1 knockdown enhances hypoxia-induced mitophagy and that expression of phosphorylation site mutants affects cellular processes like senescence . Translating these findings to therapeutic strategies requires further validation in preclinical models.

How do different fixation and preparation methods affect the performance of Phospho-BCL2L1 (S62) Antibody in immunofluorescence applications?

Fixation and preparation methods significantly impact the performance of phospho-specific antibodies in immunofluorescence applications:

• Fixation agents:

  • Paraformaldehyde (4%) is generally effective for phospho-epitope preservation

  • Methanol fixation may cause phospho-epitope loss for some antibodies but can improve access to certain antigens

  • Glutaraldehyde should typically be avoided as it can cause high background with phospho-antibodies

• Fixation timing: Rapid fixation is critical for preserving phosphorylation status, as phosphatases remain active until samples are fully fixed. For adherent cells, directly adding fixative to culture medium rather than trypsinizing can better preserve phosphorylation states.

• Permeabilization optimization:

  • Triton X-100 (0.1-0.5%) is commonly used but may extract some membrane-associated proteins

  • Gentler detergents like saponin (0.1%) might better preserve cellular architecture

  • For the recommended 1:50-1:100 dilution of Phospho-BCL2L1(S62) antibody in IF/ICC applications , optimal permeabilization is essential for antibody access to intracellular targets

• Antigen retrieval: For tissue sections or strongly fixed samples, antigen retrieval methods may be necessary:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Tris-EDTA buffer (pH 9.0) may be more effective for some phospho-epitopes

  • Enzymatic retrieval methods should be avoided as they may cleave phosphate groups

• Blocking considerations:

  • Use phosphate-free blocking agents when possible

  • BSA (3-5%) is preferred over milk-based blockers, which contain phosphoproteins and phosphatases

  • Include phosphatase inhibitors in blocking and antibody diluent buffers

• Signal amplification:

  • Tyramide signal amplification can enhance detection of low-abundance phospho-proteins

  • Fluorophore-conjugated secondary antibodies with bright, photostable dyes improve signal-to-noise ratio

  • Consider using quantum dots for multiplexed detection with minimal spectral overlap

Optimizing these parameters will help maximize specific signal while minimizing background when using Phospho-BCL2L1(S62) antibody for visualizing the subcellular localization of phosphorylated BCL2L1, particularly in studying its dynamic distribution during cell cycle progression .

What are the key database entries and molecular identifiers for BCL2L1?

For comprehensive research on BCL2L1, these molecular identifiers and database entries are essential references:

Alternative names for BCL2L1 in the literature include:

• Apoptosis regulator Bcl-X

• Apoptosis regulator Bcl-X

• Apoptosis regulator BclX

• B cell lymphoma 2 like

• Bcl-2-like protein 1

• Bcl2-L-1

• BCLX

• BclXL

• BclXs

• PPP1R52 (Protein phosphatase 1 regulatory subunit 52)

These identifiers allow researchers to access comprehensive information about BCL2L1 across multiple bioinformatics platforms, facilitating literature searches, protein structure analysis, interaction studies, and evolutionary conservation analyses .

What are the recommended resources for further study of BCL2L1 phosphorylation and its biological roles?

For researchers studying BCL2L1 phosphorylation, these resources provide valuable information and tools:

• Research Areas to Explore:

  • Cancer biology

  • Cell cycle regulation

  • Apoptosis mechanisms

  • Mitophagy and mitochondrial dynamics

  • Signal transduction

  • Metabolism

• Key Research Papers:

  • "Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation During Mitosis" (2016)

  • "The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy" (2014)

  • "The Anti-apoptotic Protein BCL2L1/Bcl-xL Is Neutralized by Pro-apoptotic PMAIP1/Noxa in Neuroblastoma" (2010)

• Experimental Models:

  • Neuroblastoma cell lines: SH-EP, LAN-1, SKNSH, SH-SY5Y, IMR-32, STA-NB1, STA-NB3, STA-NB15

  • Normal human diploid BJ cells for studying effects on cellular senescence

  • HeLa cells for mitophagy studies

• Recommended Techniques:

  • Western blotting with phospho-specific antibodies (1:500-1:2000 dilution)

  • Immunofluorescence for localization studies (1:50-1:100 dilution)

  • Co-immunoprecipitation for interaction studies

  • Cell fractionation to examine subcellular distribution

  • Cell synchronization methods to study cell cycle-dependent phosphorylation

• Useful Reagents:

  • Phospho-specific antibodies for other BCL2L1 sites (T47, T115, S49)

  • Kinase inhibitors for PLK1, p38, and CDK1 to manipulate phosphorylation

  • Phosphatase inhibitors (PhosStop™) for preserving phosphorylation during sample preparation

  • Site-specific mutants (S62A, S62D) for functional studies