GABPB2 Antibody, HRP conjugated

What is GABPB2 and why is it important in molecular research?

GABPB2 (GA binding protein transcription factor, beta subunit 2) is a protein-coding gene that functions as a transcription factor capable of interacting with purine-rich GA repeats. Its importance lies in its role in epigenetics and nuclear signaling pathways, particularly in transcriptional regulation. GO annotations related to this gene include protein homodimerization activity and transcription regulatory region DNA binding . Understanding GABPB2 function provides insights into fundamental gene expression regulatory mechanisms in human cells.

What are the optimal storage conditions for GABPB2 Antibody, HRP conjugated?

The optimal storage conditions for GABPB2 Antibody, HRP conjugated require aliquoting and storing at -20°C or -80°C. It's critical to avoid repeated freeze/thaw cycles which can degrade the antibody and reduce its effectiveness. Additionally, since the antibody is conjugated to HRP (horseradish peroxidase), it's important to avoid exposure to light which can diminish enzymatic activity . The antibody should be stored in its buffer solution (typically 0.01M PBS, pH 7.4, with 0.03% Proclin-300 and 50% Glycerol) which helps maintain stability. Proper storage ensures antibody integrity for up to 12 months.

What is the principle behind using HRP conjugation in GABPB2 antibodies?

HRP (horseradish peroxidase) conjugation to GABPB2 antibodies operates on the principle of enzyme-linked detection systems. The HRP enzyme catalyzes the oxidation of substrates (such as TMB, DAB, or luminol) in the presence of hydrogen peroxide, producing colorimetric, chemiluminescent, or fluorescent signals that can be quantified . This direct conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental workflows and potentially reducing background noise. The HRP conjugation provides enhanced sensitivity for detecting low-abundance GABPB2 protein in complex biological samples, making it particularly valuable for techniques like ELISA where signal amplification is beneficial.

What are the recommended dilution ranges for different experimental applications?

For ELISA applications with GABPB2 Antibody, HRP conjugated, the manufacturer typically recommends that optimal dilutions should be determined by the end user through titration experiments . Based on similar antibodies targeting GABPB2, typical dilution ranges for different applications include: 1:500-1:2000 for Western Blotting, 1:50-1:200 for Immunohistochemistry , and 1:100-1:500 for Immunofluorescence. These ranges serve as starting points for optimization, and researchers should establish the minimum antibody concentration that yields maximum specific signal with minimal background for their specific experimental system.

How can I validate the specificity of GABPB2 Antibody, HRP conjugated in my experimental system?

Validating the specificity of GABPB2 Antibody, HRP conjugated requires a multi-faceted approach. First, include positive controls using cell lines known to express GABPB2, such as MCF-7 or MDA-MB-231 cells, which have been verified for GABPB2 detection . Second, implement negative controls through GABPB2 knockdown/knockout systems or cell lines known to lack GABPB2 expression. Third, conduct specificity tests using competitive blocking with the recombinant GABPB2 protein (specifically amino acids 163-440, which was used as the immunogen) . Additionally, compare results with alternative GABPB2 antibodies targeting different epitopes to confirm consistent detection patterns. Finally, verify the molecular weight of detected proteins matches the expected size of approximately 49 kDa .

What sample types have been verified with GABPB2 antibodies?

GABPB2 antibodies have been verified with multiple sample types. For Western blotting, verified samples include human breast cancer cell lines such as MCF-7 and MDA-MB-231 . For immunohistochemistry applications, human colon cancer tissue and human brain tissue have been successfully used . The antibodies show reactivity with both human and mouse samples , though researchers should note that specificity may vary depending on the particular antibody clone and application. When working with novel sample types, preliminary validation is recommended, particularly for tissue-specific expression patterns or when investigating potential isoform variations.

What are common sources of background when using GABPB2 Antibody, HRP conjugated, and how can they be mitigated?

Common sources of background when using GABPB2 Antibody, HRP conjugated include: (1) Non-specific binding of the antibody, which can be reduced by optimizing antibody dilution and including appropriate blocking agents (typically 3-5% BSA or non-fat milk) in diluent buffers ; (2) Endogenous peroxidase activity in biological samples, which should be quenched using hydrogen peroxide treatment prior to antibody application; (3) Insufficient washing, requiring additional wash steps with detergent-containing buffers; (4) Cross-reactivity with similar epitopes, mitigated through antibody pre-absorption with potentially cross-reactive proteins; and (5) HRP substrate overdevelopment, controlled through timed development and stopping reactions at appropriate intervals. Additionally, endogenous biotin or avidin-binding proteins may cause interference in certain detection systems.

How can I optimize signal detection when working with low-abundance GABPB2 protein?

Optimizing signal detection for low-abundance GABPB2 protein requires a systematic approach. First, increase sample concentration through techniques like immunoprecipitation or cell fractionation to enrich for nuclear proteins, as GABPB2 is primarily localized to the nucleus . Second, implement signal amplification methods compatible with HRP, such as tyramide signal amplification (TSA), which can increase sensitivity by 10-100 fold. Third, optimize incubation conditions by extending primary antibody incubation times (overnight at 4°C) and ensuring optimal temperature for enzyme activity during detection. Fourth, select high-sensitivity substrates such as enhanced chemiluminescent (ECL) reagents for Western blotting or high-sensitivity colorimetric/fluorogenic substrates for ELISA. Finally, increase exposure times or detector sensitivity settings while monitoring background to maintain optimal signal-to-noise ratios.

What are the potential causes of false positive or false negative results when using GABPB2 Antibody, HRP conjugated?

False positive results when using GABPB2 Antibody, HRP conjugated may stem from: (1) Cross-reactivity with homologous proteins, particularly GABPB1, which shares high sequence homology with GABPB2 ; (2) Non-specific binding to highly charged or hydrophobic proteins; (3) Sample contamination with bacteria expressing protein A/G that bind immunoglobulins; or (4) Overly sensitive detection systems with improper thresholding. False negative results can occur due to: (1) Protein degradation from improper sample handling; (2) Epitope masking through protein modifications or complex formation; (3) Insufficient antigen retrieval in fixed samples; (4) HRP inactivation due to azide contamination or exposure to light; or (5) GABPB2 expression below detection threshold. Appropriate controls, including recombinant GABPB2 protein standards, are essential for distinguishing true from false results.

How should I address inconsistent results between experiments using GABPB2 Antibody, HRP conjugated?

Addressing inconsistent results between experiments requires systematic investigation of variables. First, establish standardized protocols with precise documentation of reagent preparation, incubation times/temperatures, and washing procedures to ensure experimental reproducibility. Second, implement antibody validation with each new lot using positive controls (recombinant GABPB2 protein or verified GABPB2-expressing cell lines) . Third, monitor antibody stability by avoiding repeated freeze-thaw cycles and checking for precipitation or contamination. Fourth, standardize sample preparation methods including consistent protein extraction protocols, protein quantification, and storage conditions. Fifth, normalize experimental conditions across runs by using the same equipment settings, calibrated pipettes, and consistent reagent sources. Additionally, consider biological variations in GABPB2 expression related to cell cycle stage or culture conditions that might contribute to result variability.

How can I use GABPB2 Antibody, HRP conjugated in multiplex detection systems?

Utilizing GABPB2 Antibody, HRP conjugated in multiplex detection systems requires strategic planning to avoid signal interference. For chromogenic multiplexing, pair the HRP-conjugated GABPB2 antibody with antibodies using distinct enzyme systems (such as alkaline phosphatase) that develop different colored precipitates. For fluorescent multiplexing, consider using HRP-activated tyramide signal amplification (TSA) with spectrally distinct fluorophores. Sequential detection protocols can be implemented by completely inactivating HRP after the first detection using hydrogen peroxide treatment before proceeding with the next marker. When designing multiplex panels, account for subcellular localization differences, as GABPB2 is primarily nuclear , allowing spatial separation from cytoplasmic or membrane markers. Careful optimization of antibody dilutions is essential to balance signal intensities across all markers in the multiplex panel.

What approaches can be used to study GABPB2 protein-protein interactions using GABPB2 Antibody, HRP conjugated?

To study GABPB2 protein-protein interactions using GABPB2 Antibody, HRP conjugated, several sophisticated approaches can be employed. Co-immunoprecipitation followed by proximity ligation assays (PLA) can be performed using the GABPB2 antibody paired with antibodies against suspected interaction partners. For chromatin interactions, chromatin immunoprecipitation (ChIP) using GABPB2 antibody can identify DNA binding regions, with sequential ChIP (Re-ChIP) revealing co-occupancy with other transcription factors. ELISA-based protein interaction assays can be developed using immobilized GABPB2 antibody to capture the protein complex, followed by detection with antibodies against interaction partners. Additionally, the GABPB2 Antibody, HRP conjugated can be used in protein microarrays to screen for novel interaction partners in high-throughput formats. In all these applications, appropriate controls must be included to distinguish specific from non-specific interactions.

How can I quantitatively analyze GABPB2 expression levels using GABPB2 Antibody, HRP conjugated?

Quantitative analysis of GABPB2 expression using GABPB2 Antibody, HRP conjugated can be achieved through several methodologies. For ELISA-based quantification, develop a standard curve using recombinant GABPB2 protein (specifically the immunogen region of amino acids 163-440) at known concentrations to calculate absolute GABPB2 levels in samples. For Western blot quantification, use densitometry analysis of bands together with normalization to housekeeping proteins and include recombinant GABPB2 standards. When quantifying across multiple experiments, include consistent positive control samples on each assay to allow inter-assay normalization. For tissue samples in IHC applications, employ digital image analysis using specialized software to quantify staining intensity and distribution patterns. In all quantitative applications, ensure the analysis is performed within the linear range of detection and validate that signal intensity correlates linearly with protein concentration through dilution series experiments.

What are the considerations for using GABPB2 Antibody, HRP conjugated in primary tissue samples versus cell lines?

When using GABPB2 Antibody, HRP conjugated with primary tissue samples versus cell lines, several key differences must be considered. Primary tissues present greater cellular heterogeneity requiring careful interpretation of staining patterns and potentially cell-type specific analysis. Tissue fixation methods significantly impact epitope accessibility, necessitating optimization of antigen retrieval protocols, which is less critical for cell lines. Endogenous peroxidase activity is typically higher in primary tissues (particularly blood-rich samples) requiring more rigorous quenching procedures. The nuclear localization of GABPB2 demands proper permeabilization of the nuclear membrane, which can be more challenging in certain fixed tissues. Primary tissues may express GABPB2 at lower levels than certain cell lines, potentially requiring signal amplification strategies. Additionally, autofluorescence is more pronounced in tissues (especially with formalin fixation), which may interfere with certain detection methods even when using HRP-conjugated antibodies.

What controls should be included when designing experiments with GABPB2 Antibody, HRP conjugated?

A comprehensive control strategy for experiments with GABPB2 Antibody, HRP conjugated should include: (1) Positive controls using verified GABPB2-expressing samples such as MCF-7 or MDA-MB-231 cells or human colon cancer tissue ; (2) Negative controls including isotype-matched irrelevant antibodies to assess non-specific binding; (3) Antigen blocking controls where the antibody is pre-incubated with recombinant GABPB2 protein to confirm specificity; (4) Technical negative controls omitting primary antibody to evaluate secondary reagent specificity and background; (5) Endogenous enzyme controls to assess the effectiveness of peroxidase quenching in tissue samples; (6) Biological reference controls such as GABPB2 knockdown or knockout samples if available; and (7) Dilution series of recombinant protein standards for quantitative applications. For new experimental systems, preliminary validation with multiple GABPB2 antibodies targeting different epitopes provides additional confidence in specificity.

How can I distinguish between GABPB1 and GABPB2 detection in my experiments?

Distinguishing between GABPB1 and GABPB2 detection requires careful consideration due to their high sequence homology . First, verify the epitope specificity of the GABPB2 Antibody, HRP conjugated by reviewing the immunogen sequence (amino acids 163-440 of human GABPB2) and comparing it with GABPB1 sequences to identify regions of divergence. Second, perform parallel experiments with specific antibodies against GABPB1 to compare expression patterns. Third, use molecular weight differentiation, as GABPB1 and GABPB2 have slightly different predicted molecular weights (GABPB2 is approximately 49 kDa) . Fourth, employ RNA interference techniques targeting unique regions of each transcript to selectively knock down either GABPB1 or GABPB2 and observe changes in antibody detection patterns. Finally, consider complementary nucleic acid-based methods like RT-PCR with isoform-specific primers to correlate protein detection with mRNA expression profiles.

What are the implications of GABPB2's nuclear localization for experimental design?

GABPB2's nuclear localization has several important implications for experimental design. Sample preparation protocols must ensure effective nuclear membrane permeabilization, particularly for immunofluorescence and flow cytometry applications, through optimized detergent concentrations or heat-induced epitope retrieval methods. Subcellular fractionation may be necessary to enrich for nuclear proteins before Western blotting or immunoprecipitation to improve detection sensitivity. Co-localization studies should include established nuclear markers to confirm proper nuclear staining patterns. For live-cell imaging applications, membrane-permeable substrates must be selected if using the HRP-conjugated antibody in fixed-cell protocols. Additionally, the nuclear localization suggests potential roles in transcriptional regulation, directing experimental design toward techniques like chromatin immunoprecipitation (ChIP) to identify DNA binding regions, or co-immunoprecipitation to discover interactions with other nuclear factors involved in transcriptional complexes.

How should I interpret unexpected molecular weight bands when using GABPB2 Antibody, HRP conjugated in Western blotting?

When encountering unexpected molecular weight bands in Western blotting with GABPB2 Antibody, HRP conjugated, systematic interpretation is essential. Lower molecular weight bands may represent: (1) Proteolytic degradation products, addressable by adding protease inhibitors during sample preparation; (2) Alternative splice variants of GABPB2; or (3) Cross-reactivity with related proteins. Higher molecular weight bands could indicate: (1) Post-translational modifications such as phosphorylation, ubiquitination, or SUMOylation; (2) Protein complexes resistant to denaturation; or (3) Non-specific aggregation. To distinguish between these possibilities, implement additional controls including: phosphatase treatment to identify phosphorylated forms, comparison with recombinant GABPB2 protein standards, competition experiments with blocking peptides, and validation with alternative GABPB2 antibodies targeting different epitopes. Mass spectrometry analysis of the unexpected bands can provide definitive identification. Remember that the canonical form of GABPB2 has a calculated molecular weight of approximately 49 kDa .

How can GABPB2 Antibody, HRP conjugated be utilized in cancer research models?

GABPB2 Antibody, HRP conjugated can be strategically utilized in cancer research through multiple approaches. For tumor tissue microarray analysis, the antibody can quantify GABPB2 expression across different cancer types and correlate levels with clinicopathological features and patient outcomes. In cancer cell line panels, comparative GABPB2 expression analysis can identify associations with specific oncogenic mutations or phenotypes. The antibody can be used in chromatin immunoprecipitation sequencing (ChIP-seq) experiments to map GABPB2 binding sites across the genome in normal versus cancer cells, potentially identifying dysregulated target genes. For functional studies, GABPB2 expression can be monitored during cancer-relevant processes such as epithelial-mesenchymal transition (EMT) or in response to therapeutic agents. The nuclear localization of GABPB2 suggests potential roles in transcriptional regulation of cancer-associated genes, which can be explored through correlation with known oncogenic transcription factor networks.

What considerations are important when using GABPB2 Antibody, HRP conjugated in studies of transcriptional regulation?

When using GABPB2 Antibody, HRP conjugated in transcriptional regulation studies, several specialized considerations are important. First, complement antibody-based detection with functional assays such as reporter gene assays to correlate GABPB2 binding with transcriptional outcomes. Second, design experiments to distinguish between direct and indirect regulatory effects by combining ChIP-seq data with RNA-seq after GABPB2 modulation. Third, investigate GABPB2 interactions with other transcription factors and coregulators through sequential ChIP (Re-ChIP) or proximity ligation assays. Fourth, examine how post-translational modifications affect GABPB2 function by comparing phosphorylated versus non-phosphorylated forms using phospho-specific antibodies alongside the total GABPB2 antibody. Fifth, consider the dynamics of GABPB2 nuclear localization in response to cellular signaling through time-course experiments. Finally, integrate GABPB2 binding data with chromatin accessibility profiles (ATAC-seq or DNase-seq) to understand the relationship between GABPB2 binding and chromatin state.

How do post-translational modifications affect GABPB2 detection with HRP-conjugated antibodies?

Post-translational modifications (PTMs) can significantly impact GABPB2 detection with HRP-conjugated antibodies through several mechanisms. If the antibody's epitope region (amino acids 163-440) contains sites for phosphorylation, ubiquitination, SUMOylation, or other modifications, these PTMs may either mask the epitope or alter its conformation, potentially reducing antibody binding affinity. Conversely, if the antibody was raised against a modified form of the protein, it might preferentially recognize the modified epitope over the unmodified version. PTMs can also alter GABPB2's apparent molecular weight in gel-based applications, with phosphorylation typically adding ~0.5-1 kDa per site and ubiquitination adding ~8.5 kDa per modification. To comprehensively characterize GABPB2 PTMs, researchers should consider parallel experiments with phosphatase or deubiquitinase treatments, as well as employing modification-specific antibodies alongside the total GABPB2 antibody to distinguish modified subpopulations within the total GABPB2 pool.

How can GABPB2 Antibody, HRP conjugated be integrated into emerging single-cell analysis technologies?

Integrating GABPB2 Antibody, HRP conjugated into single-cell analysis technologies represents an emerging frontier requiring innovative approaches. For mass cytometry (CyTOF) applications, metal isotope conjugation protocols can be adapted from HRP-conjugated antibodies, enabling GABPB2 detection within high-parameter single-cell protein panels. In microfluidic-based single-cell Western blotting, the HRP conjugation provides direct detection capability in nanoliter-scale protein separations. For spatial transcriptomics platforms, the antibody can be used in parallel immunofluorescence imaging following tyramide signal amplification (TSA) to correlate GABPB2 protein localization with gene expression patterns at single-cell resolution. In computational integration approaches, GABPB2 protein detection data can be aligned with single-cell RNA-seq using antibody-based cell hashing techniques. These integrative approaches will provide unprecedented insights into cellular heterogeneity of GABPB2 expression and function, particularly valuable in complex tissues where the nuclear transcription factor may play cell type-specific roles.

What methods can be used to assess the functional impact of GABPB2 binding to target genes?

Assessing the functional impact of GABPB2 binding to target genes requires a multi-faceted approach combining genomic, transcriptomic, and functional techniques. Chromatin immunoprecipitation sequencing (ChIP-seq) using GABPB2 Antibody, HRP conjugated with appropriate modifications can map genome-wide binding sites, while CUT&RUN or CUT&Tag methods offer higher resolution with lower cell input requirements. Integrating binding data with RNA-seq following GABPB2 knockdown, knockout, or overexpression reveals direct transcriptional consequences of binding. For specific target genes, reporter assays using luciferase constructs containing GABPB2 binding sites can quantify transcriptional activation or repression. Genome editing approaches like CRISPR interference (CRISPRi) targeted to GABPB2 binding sites can disrupt local interactions without removing the entire transcription factor. Finally, chromosome conformation capture techniques (4C, Hi-C) can determine how GABPB2 binding influences three-dimensional chromatin organization, potentially mediating enhancer-promoter interactions that regulate target gene expression.

How might GABPB2 protein interactions vary across different cellular contexts and disease states?

GABPB2 protein interactions likely exhibit significant context-specific variability across cellular conditions and disease states through several mechanisms. In different cell types, the available interactome may vary based on cell-specific expression patterns of potential binding partners, creating tissue-specific regulatory complexes. During cellular differentiation or activation, dynamic changes in GABPB2 post-translational modifications may alter its affinity for different binding partners. In disease contexts such as cancer, mutations in either GABPB2 or its interaction partners could disrupt normal complexes or create neomorphic interactions. Stress conditions may trigger signaling cascades that alter GABPB2 localization or conformation, switching its interaction profile. Systematic investigation of these context-dependent interactions requires affinity purification-mass spectrometry (AP-MS) using GABPB2 Antibody across multiple cell types and conditions, complemented by proximity labeling approaches such as BioID or APEX to capture transient interactions. These interaction networks can then be correlated with functional outcomes to understand how context-specific protein complexes contribute to normal function and disease mechanisms.

What technological advances might enhance the utility of GABPB2 antibodies in future research applications?

Emerging technological advances promise to significantly enhance GABPB2 antibody utility in future research. Nanobody or single-domain antibody development against GABPB2 would enable superior tissue penetration, reduced immunogenicity, and expanded applications in intracellular tracking. Site-specific conjugation technologies could replace traditional HRP labeling with precisely positioned fluorophores or enzymes, improving activity and reducing batch-to-batch variability. Antibody engineering through display technologies (phage, yeast, or mammalian) could generate higher-affinity variants with improved specificity for distinguishing between highly homologous GABPB1 and GABPB2. Bifunctional antibody formats could simultaneously target GABPB2 and known interacting partners to directly visualize protein complexes in situ. Photoswitchable or environmentally responsive antibody conjugates would enable dynamic studies of GABPB2 behavior under varying cellular conditions. Integration with emerging spatial multi-omics platforms will allow correlation of GABPB2 protein levels with transcriptomic and epigenomic features at subcellular resolution. These technological advances will collectively transform GABPB2 antibodies from static detection tools into dynamic probes for mechanistic studies.

Data Table: Comparative Analysis of GABPB2 Antibody Applications