ascl1a Antibody

What is ASCL1/ascl1a and what cellular systems express this protein?

ASCL1 (Achaete-scute complex homolog 1) is a member of the basic helix-loop-helix (bHLH) family of transcription factors. In humans, this protein is encoded by the ASCL1 gene, which may also be known as HASH1, MASH1, bHLHa46, achaete-scute homolog 1, and ASH-1 . The protein has a molecular weight of approximately 25.5 kilodaltons and activates transcription by binding to E-box motifs (5'-CANNTG-3') . ASCL1 requires dimerization with other bHLH proteins for efficient DNA binding and plays crucial roles in neuronal commitment and differentiation, particularly in the generation of olfactory and autonomic neurons . The protein is notably expressed in epithelium lobar bronchus neuroendocrine cells and lung neuroendocrine cells . In zebrafish, the orthologous gene ascl1a has been implicated in retina regeneration through regulation of Müller glia dedifferentiation .

What are the optimal dilutions and applications for ASCL1 antibodies?

Based on validated research protocols, ASCL1 antibodies perform optimally in several applications with specific dilution ranges:

It is recommended that researchers titrate antibodies in each testing system to obtain optimal results, as performance can be sample-dependent . For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 can serve as an alternative .

How should ASCL1 antibodies be stored and handled for maximum stability?

For optimal stability and performance, ASCL1 antibodies should be stored at -20°C where they typically remain stable for one year after shipment . Many commercial ASCL1 antibodies are supplied in storage buffer consisting of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain antibody integrity . For antibodies in this formulation, aliquoting is generally unnecessary for -20°C storage. Some preparations may contain 0.1% BSA, particularly in smaller volume products (20μl sizes) . When working with the antibody, avoid repeated freeze-thaw cycles and maintain cold chain practices during experiments. Always centrifuge the antibody vial briefly before opening to collect liquid that may have accumulated on the cap or sides during shipping or storage.

How can researchers validate the specificity of ASCL1 antibodies?

Validating antibody specificity is crucial for reliable experimental results. A comprehensive validation approach for ASCL1 antibodies should include:

• Positive and negative controls: Use cell lines with known ASCL1 expression (such as Y79 cells as a positive control) and compare with tissues or cell lines that do not express ASCL1.

• Knockdown/knockout validation: Perform siRNA knockdown or CRISPR-Cas9 knockout of ASCL1 and confirm reduction or absence of signal with the antibody.

• Molecular weight confirmation: Verify that the observed molecular weight matches the expected size of ASCL1 (approximately 25 kDa) .

• Cross-reactivity assessment: Test reactivity across multiple species if working with non-human models. Available antibodies show various reactivity patterns with human, mouse, and rat samples .

• Multiple antibody comparison: Use antibodies from different suppliers or those targeting different epitopes of ASCL1 to confirm consistent staining patterns.

• Peptide competition assay: Pre-incubate the antibody with a blocking peptide containing the target epitope and confirm loss of signal.

• Immunoprecipitation followed by mass spectrometry: This gold-standard approach can verify that the antibody is pulling down the correct protein.

What are the critical considerations for performing ChIP experiments with ASCL1 antibodies?

Chromatin immunoprecipitation (ChIP) with ASCL1 antibodies requires careful experimental design. Based on successful protocols:

• Cross-linking optimization: Standard formaldehyde fixation (1%) for 10 minutes at room temperature works for most applications, but optimization may be necessary depending on cell type.

• Antibody selection: Choose ChIP-validated antibodies. For tagged constructs, tag-specific antibodies (e.g., anti-Myc for Myc-Ascl1a constructs) have been successfully used .

• Control selection: Include an IgG control from the same species as the ASCL1 antibody and, if possible, input controls.

• Target region design: When designing primers for qPCR validation, focus on regions containing E-box motifs (CANNTG), particularly those similar to known ASCL1 binding sites (CAGCTG and CAGGTG) .

• Known binding sites: Previous research has identified ASCL1 binding to promoter regions containing clustered E-box motifs, such as those found in the lin-28 promoter .

• Data analysis: When analyzing ChIP-seq data, focus on regions containing the canonical E-box motifs, preferably with multiple sites clustered together, as these are more likely to represent true ASCL1 binding sites .

How can ASCL1 antibodies be utilized to study its role in cancer progression?

Recent research has revealed that ASCL1 plays important roles in cancer biology, particularly in breast cancer where it has been shown to be upregulated and associated with unfavorable prognosis . Researchers can employ ASCL1 antibodies in multiple sophisticated approaches:

• Expression profiling: IHC and tissue microarray analysis with validated ASCL1 antibodies can help correlate expression levels with clinical outcomes across patient cohorts.

• Mechanistic studies: Western blotting can detect changes in ASCL1 expression and its downstream effectors following experimental manipulations, such as drug treatments or genetic modifications.

• Pathway analysis: Co-immunoprecipitation with ASCL1 antibodies followed by mass spectrometry can identify novel binding partners in cancer-specific contexts.

• Chromatin occupancy: ChIP-seq using ASCL1 antibodies can map the cancer-specific gene regulatory networks controlled by this transcription factor.

• Therapeutic targeting assessment: ASCL1 antibodies can be used to monitor protein levels following treatment with potential inhibitors. This approach is particularly relevant given recent findings that inhibition of ASCL1 increases cancer cell sensitivity to paclitaxel both in vitro and in vivo .

• Ferroptosis studies: Immunoblotting for ASCL1 and related pathway components (CREB1, GPX4) can help elucidate the mechanism by which ASCL1 inhibition activates ferroptosis in cancer cells .

What experimental approaches can resolve contradictory results when using different ASCL1 antibodies?

When faced with contradictory results using different ASCL1 antibodies, researchers should implement a systematic troubleshooting approach:

• Epitope mapping comparison: Determine which region of ASCL1 each antibody targets (N-terminal, C-terminal, or internal domains) . Epitope availability may differ depending on experimental conditions, protein modifications, or binding partners.

• Validation in multiple systems: Test antibodies in well-characterized positive control systems, such as Y79 cells for Western blotting or human brain tissue for IHC .

• Antibody characterization: Compare monoclonal versus polyclonal antibodies. Polyclonal antibodies recognize multiple epitopes and may provide more robust detection but potentially with higher background, while monoclonals offer higher specificity but might miss certain protein isoforms or post-translationally modified forms .

• Orthogonal validation: Confirm results using non-antibody-based techniques such as RNA-seq, qRT-PCR, or mass spectrometry.

• Genetic validation: Perform antibody testing in systems with ASCL1 knockdown, knockout, or overexpression to definitively determine antibody specificity.

• Standardized protocols: Establish identical experimental conditions when comparing antibodies, including sample preparation, blocking reagents, incubation times, and detection methods.

• Meta-analysis: Review published literature where multiple antibodies have been compared, looking for patterns in antibody performance across different applications.

How can researchers optimize immunoprecipitation protocols for studying ASCL1's binding partners?

Optimizing immunoprecipitation (IP) for ASCL1 requires careful consideration of several factors:

• Buffer selection: For transcription factors like ASCL1, use buffers that maintain nuclear protein interactions while effectively lysing cells. RIPA buffer may be too harsh for maintaining some protein-protein interactions, while NP-40 or Triton X-100 based buffers (0.1-0.5%) may better preserve complexes.

• Antibody selection: Choose antibodies validated for IP applications, such as the Rabbit anti-ASCL1/MASH1 Recombinant Monoclonal Antibody [BLR164J] which has been validated for IP .

• Pre-clearing step: Implement a pre-clearing step with protein A/G beads to reduce non-specific binding.

• Cross-linking considerations: For transient interactions, consider using reversible cross-linking agents before cell lysis to stabilize protein complexes.

• Bead selection: Compare results using different types of beads (magnetic versus agarose) and protocols (direct versus indirect IP).

• Elution conditions: Optimize elution conditions to efficiently release ASCL1 complexes while minimizing co-elution of antibody chains, which can interfere with downstream mass spectrometry analysis.

• Controls: Always include appropriate negative controls (isotype-matched IgG) and positive controls (input lysate) in IP experiments.

• Verification: Confirm successful IP by immunoblotting a small fraction of the IP sample for ASCL1 before proceeding to protein complex analysis.

What are the key differences in using ASCL1 antibodies across various species models?

When working with ASCL1 antibodies across different model organisms, researchers should consider several species-specific factors:

How can ASCL1 antibodies be used to study neuronal differentiation mechanisms?

ASCL1 plays a critical role in neuronal differentiation, making antibodies against this protein valuable tools for neurodevelopmental research:

• Temporal expression analysis: Track ASCL1 expression during different stages of neuronal differentiation using immunofluorescence or Western blotting. This approach can reveal critical temporal windows when ASCL1 influences cell fate decisions.

• Lineage tracing: Combine ASCL1 immunostaining with other neural lineage markers to determine which neural subtypes derive from ASCL1-expressing progenitors.

• Reprogramming studies: Monitor ASCL1 levels during direct neuronal reprogramming of non-neural cells, correlating expression with conversion efficiency and neuronal subtype specification.

• ChIP-seq applications: Use ASCL1 antibodies for ChIP-seq to identify downstream genetic programs activated during neuronal differentiation. Focus on E-box containing promoters (CANNTG), particularly those with consensus sequences similar to known ASCL1 binding sites (CAGCTG and CAGGTG) .

• Protein complex analysis: Perform co-immunoprecipitation with ASCL1 antibodies at different differentiation stages to identify stage-specific binding partners that may modulate its function.

• Subcellular localization: Use immunofluorescence with ASCL1 antibodies to track protein localization during differentiation, as nuclear translocation often correlates with transcriptional activity.

• Post-translational modifications: Combine ASCL1 immunoprecipitation with mass spectrometry to identify differentiation stage-specific post-translational modifications that may regulate its activity.

How can researchers use ASCL1 antibodies to investigate the CREB1/GPX4 axis in ferroptosis?

Recent research has uncovered a novel role for ASCL1 in regulating ferroptosis through the CREB1/GPX4 axis, particularly in breast cancer . Researchers can employ the following methods using ASCL1 antibodies:

• Signaling pathway analysis: Use Western blotting with phospho-specific antibodies to track CREB1 phosphorylation status following ASCL1 modulation. Current evidence indicates that inhibition of ASCL1 decreases CREB1 phosphorylation, subsequently reducing GPX4 expression .

• Chromatin occupancy studies: Perform ChIP-seq with ASCL1 antibodies to identify potential direct binding to regulatory regions of CREB1 or GPX4, or to shared target genes that influence ferroptosis pathways.

• Double immunofluorescence: Co-stain for ASCL1 and GPX4 to determine spatial relationships in cellular systems and tissue samples, particularly following treatments that modulate ferroptosis.

• Protein-protein interaction studies: Use co-immunoprecipitation with ASCL1 antibodies followed by immunoblotting for CREB1 to investigate whether these proteins form complexes that could directly regulate gene expression.

• Functional validation: Combine ASCL1 immunoblotting with ferroptosis markers (MDA, ROS, GSH levels, GSH/GSSG ratio) and mitochondrial morphology analysis to correlate ASCL1 levels with ferroptotic events .

• Therapeutic targeting assessment: Monitor ASCL1, CREB1, and GPX4 levels using validated antibodies when testing potential ferroptosis inducers or inhibitors, particularly in cancer models where ASCL1 inhibition has been shown to increase paclitaxel sensitivity .

What approaches can resolve contradictory data between ASCL1 protein levels and cellular phenotypes?

When faced with discrepancies between ASCL1 protein detection and expected cellular phenotypes, researchers should consider these methodological approaches:

• Context-dependent activity: Assess ASCL1 in conjunction with known co-factors, as its activity is highly dependent on dimerization with other bHLH proteins . Co-immunoprecipitation followed by mass spectrometry can identify relevant binding partners in specific cellular contexts.

• Post-translational modification analysis: Investigate whether ASCL1 is subject to modifications that affect its activity but not detection. Phosphorylation, ubiquitination, or other modifications may alter function without significantly changing antibody recognition.

• Functional redundancy: Examine expression of related bHLH family members that may compensate for ASCL1 in certain contexts, potentially explaining why phenotypes don't always correlate with ASCL1 levels.

• Antibody epitope accessibility: Test multiple antibodies targeting different regions of ASCL1 to ensure that all protein forms are being detected. Some antibodies specifically target the C-terminal region , which may be masked in certain protein complexes.

• Subcellular localization: Use fractionation followed by immunoblotting or immunofluorescence to determine whether ASCL1 localization, rather than total levels, correlates with phenotypes.

• Threshold effects: Establish dose-response relationships through careful titration experiments to determine whether certain phenotypes require threshold levels of ASCL1 that may not be linearly related to total protein.

• Temporal dynamics: Implement time-course experiments to capture dynamic changes in ASCL1 levels that might be missed in single time-point analyses but could explain phenotypic outcomes.