MEIS3 Antibody, Biotin conjugated

What is MEIS3 and what biological functions have been identified for this protein?

MEIS3 (Meis Homeobox 3) functions as a transcriptional regulator that directly modulates PDPK1 expression, thus promoting survival of pancreatic beta-cells. Additionally, MEIS3 regulates expression of several other genes including NDFIP1, BNIP3, and CCNG1 . As a member of the TALE (three amino acid loop extension) homeodomain family, MEIS3 is involved in chromatin binding pathways and plays important roles in transcriptional regulation . The protein contains multiple functional domains, with the antibody described in the search results targeting the region spanning amino acids 178-264 .

What are the technical advantages of using biotin-conjugated antibodies for MEIS3 detection compared to unconjugated alternatives?

Biotin-conjugated antibodies offer several significant advantages for MEIS3 detection. The biotin-streptavidin interaction provides one of the strongest non-covalent biological binding systems known, enabling high sensitivity detection protocols. When using biotin-conjugated MEIS3 antibodies, researchers benefit from signal amplification capabilities, as multiple streptavidin molecules (conjugated to detection systems) can bind to each biotinylated antibody . Additionally, these conjugated antibodies enable flexible experimental design, allowing researchers to choose from various detection systems (fluorescent, enzymatic, etc.) without changing the primary antibody. For MEIS3 detection specifically, biotin-conjugated antibodies facilitate sensitive detection in applications such as ELISA, where they have demonstrated high specificity .

What experimental applications are biotin-conjugated MEIS3 antibodies most suitable for?

Biotin-conjugated MEIS3 antibodies are particularly well-suited for ELISA applications, as explicitly stated in the product specifications . Beyond this primary application, these antibodies can be effectively utilized in several additional techniques. They enable efficient enrichment of biotinylated peptides through anti-biotin antibody-based pull-down methods, which is useful in protein interaction studies . They can also be employed in specialized proximity-dependent biotinylation approaches to study MEIS3 protein complexes . The biotin conjugation allows for signal amplification in immunohistochemistry applications, increasing detection sensitivity for low-abundance transcription factors like MEIS3. When working with biotin-conjugated MEIS3 antibodies, researchers should optimize protocol conditions specifically for the application being used.

How should researchers optimize protocol conditions when using biotin-conjugated MEIS3 antibodies in ELISA applications?

For optimal ELISA performance with biotin-conjugated MEIS3 antibodies, researchers should empirically determine the optimal working dilution as recommended by manufacturers . A methodical approach involves:

• Antibody titration: Test a range of dilutions (typically 1:500 to 1:5000) to identify the optimal signal-to-noise ratio

• Buffer optimization: Use a preservative-free blocking buffer to prevent interference with the biotin-streptavidin interaction

• Detection system selection: Employ streptavidin conjugated to HRP or other detection enzymes at appropriately optimized concentrations

• Incubation conditions: Test both temperature (4°C, room temperature) and duration variables

• Washing stringency: Implement sufficient washing steps to reduce background while preserving specific signal

The product documentation indicates that MEIS3 antibody (ABIN7155791) is preserved in 0.03% Proclin 300 with 50% glycerol in PBS (pH 7.4) , which should be considered when designing compatible buffer systems for the assay.

What strategies can researchers implement to validate the specificity of biotin-conjugated MEIS3 antibodies?

Rigorous validation of biotin-conjugated MEIS3 antibodies requires a multi-faceted approach:

This comprehensive validation approach ensures that the observed signals genuinely represent MEIS3 protein rather than non-specific binding or cross-reactivity issues .

How can researchers effectively implement biotinylation site mapping approaches to study MEIS3 protein interactions?

Biotinylation site mapping represents a powerful approach for studying MEIS3 protein interactions through specialized methodologies:

• BioSITe (Biotinylation Site Identification Technology) implementation: This approach uses anti-biotin antibodies for efficient enrichment of biotinylated peptides, avoiding the limitations of streptavidin-based capture methods . For MEIS3 interaction studies, researchers should optimize trypsin digestion conditions to generate appropriately sized peptides containing biotinylated lysines.

• Proximity-dependent biotinylation optimization: When using BirA fusion constructs or similar approaches, biotin concentration should be carefully titrated, as supplementation with 5-50 μM biotin has been demonstrated to be effective, while excessive concentrations (500 μM) may inhibit detection .

• Mass spectrometry analysis: Researchers should implement detection of signature fragment ions characteristic of biotinylated peptides, particularly ImKbio-NH3 ions which typically show median intensity of 75% of base peak . High-resolution instruments with HCD fragmentation provide optimal results for biotinylated peptide identification.

• Controls implementation: Include appropriate negative controls (non-biotinylated samples) and positive controls (known biotinylated proteins) to establish signal specificity and threshold parameters.

What common technical issues arise when working with biotin-conjugated MEIS3 antibodies, and how can they be resolved?

Researchers frequently encounter several challenges when working with biotin-conjugated MEIS3 antibodies:

• Endogenous biotin interference: Biological samples often contain endogenous biotin that competes with biotinylated antibodies for streptavidin binding. Solution: Implement avidin/biotin blocking steps prior to antibody application and consider using biotin-free media for cell culture experiments.

• Signal-to-noise optimization: Biotin-conjugated antibodies may generate high background if detection conditions are not properly optimized. Solution: Increase blocking stringency, optimize antibody concentration through titration experiments, and include appropriate negative controls.

• Storage stability issues: The documentation for MEIS3 antibody (ABIN7155791) recommends storage at -20°C or -80°C to maintain activity . Solution: Aliquot antibody upon receipt to minimize freeze-thaw cycles and follow manufacturer-recommended storage conditions.

• Preservative effects: The presence of ProClin in the antibody formulation (as specified for ABIN7155791) requires careful handling due to its hazardous properties . Solution: Always handle with appropriate safety precautions and consider potential interference effects in sensitive applications.

• Cross-reactivity concerns: Ensuring the specificity of the antibody for MEIS3 versus related homeobox proteins. Solution: Validate using the methods outlined in question 2.2, particularly with known positive and negative control samples.

How should researchers approach optimizing the sensitivity of detection when using biotin-conjugated MEIS3 antibodies?

Optimizing detection sensitivity for biotin-conjugated MEIS3 antibodies involves systematic evaluation of several experimental parameters:

• Signal amplification strategies: Implement multi-layer detection systems (biotinylated antibody → streptavidin-HRP → enhanced chemiluminescence substrates) for maximum sensitivity in Western blot and ELISA applications.

• Sample preparation optimization: For MEIS3 detection, ensure efficient nuclear protein extraction since MEIS3 functions as a transcription factor. Standard whole-cell lysates may dilute the target protein and reduce detection sensitivity.

• Incubation condition refinement: Extended incubation times at lower temperatures (e.g., overnight at 4°C) often improve binding efficiency while maintaining antibody stability and specificity.

• Detection system selection: Choose detection reagents with demonstrated low background properties. For fluorescence-based detection, select fluorophores with high quantum yield and photostability characteristics.

• Substrate optimization: For enzymatic detection systems, compare standard and enhanced sensitivity substrates to determine the optimal formulation for the specific application.

The empirical optimization of these parameters should follow a systematic approach with appropriate controls to distinguish specific signal enhancement from non-specific background increases.

What considerations are important when implementing biotin-conjugated MEIS3 antibodies in proximity-dependent biotinylation approaches?

When using biotin-conjugated MEIS3 antibodies in proximity-dependent biotinylation studies, researchers should address several methodological considerations:

• Biotin concentration optimization: Research demonstrates that supplementation with 5-50 μM biotin is generally effective for proximity labeling applications, while excessive concentrations (500 μM) may inhibit detection of biotinylated proteins like PLZF in HEK293T cells .

• Background control: Implement appropriate negative controls including non-targeting antibodies and samples without proximity labeling enzymes to establish background biotinylation levels.

• Extraction buffer selection: For subsequent analyses, 8M urea buffer has been successfully used for lysing cells in proximity biotinylation studies, ensuring efficient protein extraction while maintaining biotinylation integrity .

• Peptide identification strategies: When analyzing biotinylated peptides by mass spectrometry, utilize optimized fragmentation conditions to detect characteristic signature fragment ions of biotinylated peptides, which exhibit specific mass spectral features that aid in identification .

• Enrichment approach selection: Consider using anti-biotin antibody-based enrichment rather than streptavidin-based capture, as this approach has demonstrated efficient and reproducible enrichment of biotinylated peptides for high-throughput identification .

How can biotin-conjugated MEIS3 antibodies be integrated into multi-parameter analytical workflows?

Integration of biotin-conjugated MEIS3 antibodies into sophisticated multi-parameter analytical workflows enables comprehensive analysis of transcriptional regulation mechanisms:

• Chromatin immunoprecipitation sequencing (ChIP-seq): Biotin-conjugated MEIS3 antibodies can be employed for chromatin immunoprecipitation followed by enrichment using streptavidin beads, allowing genome-wide mapping of MEIS3 binding sites. Integration with RNA-seq data enables correlation between binding events and transcriptional outcomes.

• Sequential ChIP approaches: Biotinylated MEIS3 antibodies facilitate sequential ChIP protocols, where the streptavidin-biotin interaction is used for the first immunoprecipitation step, followed by a second IP with antibodies against potential co-factors, enabling assessment of co-occupancy at specific genomic loci.

• Proximity-dependent biotinylation integration: As demonstrated in research using AirID-based proximity labeling , biotinylated antibodies can be combined with proximity labeling approaches to identify protein-protein interaction networks in living cells.

• Single-cell analytical platforms: Biotin-conjugated antibodies enable integration with single-cell methodologies through streptavidin-based detection systems, allowing correlation of MEIS3 expression with cell-type specific transcriptional programs.

• Spatial transcriptomics: Combining in situ hybridization for MEIS3 target genes with immunodetection using biotin-conjugated MEIS3 antibodies enables spatial correlation between transcription factor localization and target gene expression.

How can researchers apply biotin-conjugated MEIS3 antibodies to investigate its role in regulating PDPK1 expression and pancreatic beta-cell survival?

The relationship between MEIS3 and PDPK1 expression in pancreatic beta-cell survival represents an important research area that can be approached using biotin-conjugated MEIS3 antibodies:

• Chromatin immunoprecipitation: Biotin-conjugated MEIS3 antibodies enable efficient ChIP to determine MEIS3 binding at the PDPK1 promoter and enhancer regions. The biotinylated antibody approach allows stringent washing conditions while maintaining specific enrichment of MEIS3-bound chromatin regions.

• Co-immunoprecipitation studies: Researchers can investigate protein complexes formed by MEIS3 at the PDPK1 locus using pull-down approaches with biotin-conjugated antibodies followed by mass spectrometry to identify co-factors that modulate MEIS3-dependent transcriptional regulation.

• Proximity labeling applications: Expression of MEIS3 fused to promiscuous biotin ligases in pancreatic beta-cells, followed by detection using biotin-conjugated MEIS3 antibodies, enables mapping of the local protein environment around MEIS3 during normal function and stress conditions.

• Functional validation studies: Combining MEIS3 knockdown/overexpression with PDPK1 expression analysis using biotin-conjugated antibodies for detection enables quantitative assessment of the regulatory relationship between these factors.

• Translational investigations: Biotin-conjugated MEIS3 antibodies facilitate examination of MEIS3 expression and localization in pancreatic tissue samples from diabetes models, allowing correlation with PDPK1 expression and beta-cell survival metrics.

What are the most effective mass spectrometry approaches for analyzing biotinylated peptides in MEIS3 interaction studies?

Mass spectrometry analysis of biotinylated peptides in MEIS3 interaction studies requires specialized approaches to ensure optimal identification and characterization:

• Fragmentation strategy optimization: Higher-energy collisional dissociation (HCD) generates characteristic biotin fragment ions that facilitate identification of biotinylated peptides. Research has identified signature fragment ions including ImKbio-NH3, which typically shows median ion intensity of 75% that of base peak from identified lysine biotinylated peptides .

• Instrument configuration: High-resolution instruments like Orbitrap Fusion Lumos mass spectrometers using a high-high acquisition mode with HCD fragmentation have been successfully employed for analyzing biotinylated peptides in proximity labeling studies .

• Enrichment methodology selection: Anti-biotin antibody-based enrichment has demonstrated advantages over streptavidin-based capture for biotinylation site mapping, providing efficient and reproducible enrichment of biotinylated peptides for high-throughput identification .

• Data analysis parameters: Implement specific search parameters that account for the mass shift corresponding to biotin addition, along with variable modifications including oxidation and deamidation that commonly occur in sample processing.

• Quantitative approaches: Consider implementing SILAC or TMT labeling strategies to enable quantitative comparison of MEIS3 interaction partners under different experimental conditions, enhancing the biological relevance of identified interactions.

How can researchers apply biotin-conjugated MEIS3 antibodies to investigate its role in chromatin binding and transcriptional regulation?

Investigating MEIS3's role in chromatin binding and transcriptional regulation can be accomplished through several advanced methodological approaches using biotin-conjugated antibodies:

• CUT&RUN or CUT&Tag methodologies: These techniques allow precise mapping of transcription factor binding sites with lower background than traditional ChIP. Biotin-conjugated MEIS3 antibodies can be employed in these protocols, with the biotin tag enabling efficient capture and detection of antibody-bound chromatin fragments.

• Chromatin conformation capture integration: Combining 3C-based techniques with MEIS3 immunoprecipitation using biotin-conjugated antibodies enables analysis of how MEIS3 binding influences three-dimensional chromatin organization, potentially identifying long-range regulatory interactions.

• In vitro DNA binding assays: Biotin-conjugated MEIS3 antibodies can be used in electrophoretic mobility shift assays (EMSA) or DNA pulldown experiments to characterize MEIS3 binding specificity and affinity for target sequences in a controlled system.

• Proteomics of isolated chromatin segments (PICh): This approach allows identification of proteins associated with specific genomic loci. Biotin-conjugated MEIS3 antibodies can be used to investigate which protein complexes are recruited to MEIS3-bound chromatin regions.

• Live-cell imaging applications: When combined with appropriate detection systems, biotin-conjugated MEIS3 antibodies enable visualization of MEIS3 dynamics in living cells, providing insights into the kinetics of chromatin binding and transcriptional activation.

What emerging technologies are expanding the applications of biotin-conjugated antibodies in transcription factor research?

Biotin-conjugated antibodies, including those targeting MEIS3, are increasingly being integrated into innovative technologies that expand their research applications. Proximity-dependent biotinylation approaches like BioID, TurboID, and AirID represent significant advances for studying protein-protein interactions in living cells . These techniques enable mapping of local protein environments around MEIS3 in its native cellular context. Additionally, the BioSITe (Biotinylation Site Identification Technology) methodology demonstrates the effective use of anti-biotin antibodies for efficient enrichment of biotinylated peptides, providing superior performance compared to traditional streptavidin-based approaches for biotinylation site mapping .

The integration of biotin-conjugated antibodies with single-cell technologies and spatial transcriptomics approaches is creating new opportunities to correlate transcription factor localization with gene expression patterns at unprecedented resolution. As these methodologies continue to evolve, biotin-conjugated MEIS3 antibodies will likely play increasingly important roles in deciphering the complex mechanisms of transcriptional regulation in normal development and disease states.