ABT1 Antibody

What is ABT1 and why is it significant in cellular research?

ABT1 (Activator of Basal Transcription 1) is a protein that plays a crucial role in gene expression regulation by associating with TATA-binding protein (TBP) to enhance basal transcription activity of class II promoters. This interaction facilitates transcription complex assembly necessary for gene activation in the nucleus . ABT1's ability to enhance transcription is particularly important in cellular processes such as growth and differentiation, where precise gene regulation is essential. Additionally, recent research has identified ABT1 as the first disease-modifying gene for SMARD1 (Spinal Muscular Atrophy with Respiratory Distress type 1), significantly increasing lifespan and decreasing neuromuscular junction denervation in mouse models .

What applications are ABT1 antibodies validated for in laboratory research?

ABT1 antibodies have been validated for multiple research applications:

These applications have been successfully employed to detect ABT1 in various species including human, mouse, rat, and other mammals . It's recommended that researchers titrate antibodies in their specific testing systems to obtain optimal results .

How can I optimize Western blot protocols for detecting endogenous ABT1 protein?

For optimal Western blot detection of endogenous ABT1:

• Lysate preparation: Use RIPA buffer with protease inhibitors for total protein extraction from tissues or cells. ABT1 has been successfully detected in mouse liver tissue and HeLa cells .

• Sample loading: Load 20-40 μg of total protein per lane for cell lysates, or 50-60 μg for tissue samples.

• Gel selection: Use 10-12% polyacrylamide gels as ABT1's observed molecular weight is 31-32 kDa .

• Transfer conditions: Transfer to PVDF membranes at 100V for 60-90 minutes in standard transfer buffer.

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute ABT1 antibody 1:200-1:1000 in blocking solution and incubate overnight at 4°C .

• Detection: Use appropriate secondary antibody and chemiluminescence detection system.

• Controls: Include positive controls (e.g., HeLa cell lysate) where ABT1 expression has been confirmed .

What are the key considerations for immunoprecipitation experiments using ABT1 antibodies?

When designing immunoprecipitation experiments with ABT1 antibodies:

• Buffer selection: Use mild lysis buffers (e.g., NP-40 or CHAPS-based) to preserve protein-protein interactions, especially when studying ABT1's interactions with binding partners like IGHMBP2 or TBP .

• Antibody selection: Choose antibodies validated for IP applications. For instance, mouse monoclonal ABT1 Antibody (B-9) has been validated for immunoprecipitation .

• Pre-clearing: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Co-IP conditions: When investigating ABT1's interactions with other proteins (e.g., IGHMBP2), use 2-5 μg of antibody per 500 μg of protein lysate .

• Controls: Include:

  • IgG control IP (same species as ABT1 antibody)

  • Input sample (5-10% of lysate used for IP)

  • Reverse IP (using antibody against predicted interacting partner)

• Washing stringency: Use increasingly stringent washes to remove non-specific interactions while preserving authentic interactions.

• Detection: For detecting ABT1 in immunoprecipitated samples, Western blotting with a different ABT1 antibody (recognizing a different epitope) can provide more specific confirmation .

How can ABT1 antibodies be used to investigate ABT1's role in modifying SMARD1 pathology?

Recent research has identified ABT1 as the first disease-modifying gene for SMARD1. To investigate this relationship using ABT1 antibodies:

• Protein interaction studies: Use co-immunoprecipitation with ABT1 antibodies to pull down and analyze IGHMBP2 complexes in relevant cell types or tissues. Research shows that ABT1 directly associates with IGHMBP2 with high binding affinity .

• Biochemical activity assays: After confirming ABT1-IGHMBP2 interaction, assess:

  • ATPase activity - Purified ABT1 significantly increases IGHMBP2's ATPase activity by approximately 1.31-fold (p < 0.0001) .

  • Helicase activity - ABT1 enhances IGHMBP2's helicase activity by approximately 1.41-fold, with most significant duplex resolution occurring when IGHMBP2 was incubated with 100 nM ABT1 .

  • Rate of DNA substrate unwinding - ABT1 association increased IGHMBP2's unwinding rate from 0.005/min to 0.01/min (1.84-fold increase, p < 0.00004) .

• RNA binding studies: Employ thermophoresis assays with and without ABT1 to study IGHMBP2's RNA binding capacity. Research indicates that ABT1 binding induces a conformational change in IGHMBP2 that exposes an RNA binding site, dramatically increasing binding affinity (KD = 1 nM when IGHMBP2, ABT1, U3 snoRNA, and scramble RNA are combined) .

• In vivo validation: Use AAV-mediated ABT1 overexpression in SMARD1 mouse models and evaluate protein levels in tissues with ABT1 antibodies to correlate with phenotypic improvements.

What are the technical considerations for using ABT1 antibodies in evaluating ABT1's role in transcriptional regulation?

For investigating ABT1's role in transcriptional regulation:

• Chromatin immunoprecipitation (ChIP):

  • Use ABT1 antibodies to identify genomic regions where ABT1 is bound.

  • Optimize crosslinking time (typically 10-15 minutes with 1% formaldehyde).

  • Ensure antibody specificity through validation with known ABT1-binding regions.

  • Include appropriate controls (IgG control, input DNA, positive control regions).

• Co-immunoprecipitation with transcription factors:

  • ABT1 associates with TBP in HeLa nuclear extracts . Design co-IP experiments to investigate this interaction in your system of interest.

  • ABT1 also interacts with Ets2 Repressor Factor (ERF) , which can be included in multifactor complex analysis.

• Functional transcription assays:

  • Combine ABT1 knockdown/overexpression with reporter gene assays to assess functional impact.

  • Use ABT1 antibodies to confirm protein levels before and after manipulation.

  • Measure transcription of known target genes after ABT1 modulation.

• Subcellular localization studies:

  • Use immunofluorescence with ABT1 antibodies (dilution 1:200-1:800) to track nuclear localization.

  • Combine with markers for transcription factories or specific promoter regions.

  • Consider co-localization with TBP and RNA polymerase II to assess functional complexes.

What are common issues in ABT1 antibody applications and how can they be resolved?

How can I validate ABT1 antibody specificity for my research applications?

Comprehensive validation of ABT1 antibody specificity should include:

• Genetic approaches:

  • Knockout/knockdown validation: Compare antibody signal in wild-type vs. ABT1 knockdown/knockout samples

  • Overexpression validation: Test detection of overexpressed ABT1 (tagged or untagged)

• Biochemical validation:

  • Western blot analysis: Confirm single band at expected molecular weight (31-32 kDa)

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Cross-reactivity assessment: Test in multiple species if working with non-human models

• Orthogonal approach validation:

  • Compare results with multiple ABT1 antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

  • Confirm subcellular localization matches known ABT1 distribution patterns

• Application-specific validation:

  • For IP applications: Verify enrichment by Western blot and mass spectrometry

  • For IF/IHC: Compare with published localization patterns and include positive controls (e.g., HeLa cells have confirmed ABT1 expression)

How can ABT1 antibodies be employed to investigate ABT1's potential role in RNA processing?

Research suggests ABT1 may modify IGHMBP2 activity as a means to regulate pre-rRNA processing . To investigate this function:

• RNA immunoprecipitation (RIP):

  • Use ABT1 antibodies to immunoprecipitate ABT1-RNA complexes

  • Analyze bound RNAs by RT-qPCR or sequencing to identify specific RNA targets

  • Compare RNA profiles between wild-type and disease models (e.g., SMARD1)

• Nucleolar co-localization studies:

  • Perform immunofluorescence with ABT1 antibodies and nucleolar markers

  • Analyze co-localization under normal conditions and stress conditions

  • Quantify changes in nucleolar localization after treatments affecting rRNA processing

• In vitro RNA processing assays:

  • Use purified ABT1 and ABT1-depleted nuclear extracts to assess pre-rRNA processing

  • Compare processing efficiency with and without ABT1

  • Analyze processing intermediates by Northern blotting

• Proximity ligation assay (PLA):

  • Use ABT1 antibodies in combination with antibodies against RNA processing factors

  • Quantify interaction frequency in different cell types and conditions

  • Correlate with rRNA processing efficiency

What approaches can be used to develop and validate bispecific antibodies involving ABT1 for research applications?

For developing bispecific antibodies (bsAbs) involving ABT1:

• Design considerations:

  • Select appropriate molecular geometry (symmetric or asymmetric)

  • Consider the intended mechanism of action and required functionality

  • Determine whether to include an Fc region based on desired properties

• Construction strategies:

  • IgG-like bsAbs: Ensure proper heavy chain and light chain pairing

  • Fragment-based approaches: Consider scFv-based or single-domain antibody formats

  • Post-expression assembly: Express individual components and assemble final bsAb through controlled redox conditions

• Validation techniques:

  • Binding specificity: Confirm binding to both ABT1 and second target using ELISA, SPR, etc.

  • Structural integrity: Analyze by size-exclusion chromatography, mass spectrometry

  • Functional activity: Verify dual functionality in relevant biological assays

• Developability assessment:

  • Biophysical characterization: Thermal stability, aggregation propensity

  • Expression yield optimization: Test different expression systems

  • Purification strategy development: Exploit differential binding properties

• Advanced characterization:

  • Conduct epitope mapping to ensure preserved binding sites

  • Analyze binding kinetics to both targets

  • Evaluate effects of potential chain mispairing on specificity and function

How can machine learning approaches be integrated with ABT1 antibody research for enhanced experimental design?

Recent advances in machine learning can be applied to ABT1 antibody research:

• Binding prediction models:

  • Use active learning algorithms to predict ABT1-antibody binding characteristics

  • Recent research shows active learning can reduce required antigen mutant variants by up to 35% and speed up the learning process compared to random baseline approaches

  • Apply these models to design optimized ABT1 antibodies with enhanced specificity

• Epitope mapping optimization:

  • Implement computational approaches to predict optimal epitopes for new ABT1 antibody development

  • Validate predictions experimentally using techniques like hydrogen-deuterium exchange mass spectrometry

  • Feed experimental data back into the model to improve future predictions

• Image analysis in microscopy applications:

  • Apply deep learning algorithms to analyze ABT1 localization patterns from immunofluorescence data

  • Identify subtle changes in distribution patterns that may correlate with functional states

  • Automate quantification of co-localization with interaction partners like IGHMBP2

• Experimental design optimization:

  • Use machine learning to identify optimal conditions for specific applications (antibody concentration, incubation time, buffer composition)

  • Implement library-on-library screening approaches with active learning to efficiently map ABT1 interaction networks

  • Develop out-of-distribution prediction models to extend findings to untested experimental conditions

What are the considerations for using ABT1 antibodies in studying post-translational modifications that may affect ABT1 function?

To investigate post-translational modifications (PTMs) of ABT1:

• PTM-specific antibody approaches:

  • Determine if commercially available ABT1 antibodies recognize PTM-modified forms

  • Consider developing modification-specific antibodies (phospho-ABT1, acetyl-ABT1, etc.)

  • Use pan-ABT1 antibodies for initial immunoprecipitation followed by PTM-specific detection

• Mass spectrometry workflow:

  • Immunoprecipitate ABT1 using validated antibodies

  • Analyze by LC-MS/MS to identify PTMs

  • Compare PTM profiles between normal and disease states or different cellular conditions

• Functional impact assessment:

  • Correlate identified PTMs with ABT1's interaction with IGHMBP2

  • Determine if PTMs affect ABT1's ability to enhance IGHMBP2's ATPase and helicase activities

  • Investigate whether PTMs regulate ABT1's association with TBP and its transcriptional activation function

• PTM dynamics analysis:

  • Study temporal changes in ABT1 PTMs following cellular stimuli

  • Identify the enzymes responsible for adding/removing PTMs

  • Develop tools to monitor PTM status in real-time in living cells

• Disease relevance investigation:

  • Compare ABT1 PTM profiles between normal individuals and SMARD1 patients

  • Determine if PTM alterations contribute to ABT1's role as a disease modifier

  • Assess potential for therapeutic targeting of specific PTMs