KHDRBS2 Antibody

What criteria should researchers consider when selecting a KHDRBS2 antibody for experimental use?

When selecting a KHDRBS2 antibody, researchers should evaluate several critical parameters to ensure optimal experimental outcomes. First, consider the specific epitope recognition—antibodies targeting different regions of KHDRBS2 may yield varying results depending on protein conformation, post-translational modifications, and protein-protein interactions. For instance, some commercially available antibodies recognize amino acids 30-79, while others target the C-terminal region or amino acids 200 to the C-terminus . Second, assess species reactivity and cross-reactivity profiles—certain antibodies demonstrate broad cross-reactivity across species (human, mouse, cow, dog, etc.) while others are more species-restricted, which is crucial for comparative studies . Third, evaluate clonality—polyclonal antibodies like ABIN6745505 and CAB6102 offer broader epitope recognition but potentially higher background, while monoclonal antibodies provide higher specificity but might be affected by epitope masking . Fourth, consider validated applications—confirm the antibody has been validated for your intended applications such as Western blotting, immunohistochemistry, ELISA, or immunofluorescence with appropriate dilution recommendations .

How can researchers validate the specificity of KHDRBS2 antibodies for their experimental systems?

Validating KHDRBS2 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. Begin with positive control validation using cell lines known to express KHDRBS2, such as HeLa, 293T, Jurkat, or K-562 cells as documented in antibody specifications . Implement knockdown/knockout validation by comparing antibody reactivity in control versus KHDRBS2-depleted samples (using siRNA, shRNA, or CRISPR methods) to confirm signal specificity, as demonstrated in studies examining KHDRBS2 function in endothelial cells . Perform peptide competition assays where pre-incubation of the antibody with excess immunogenic peptide should eliminate specific binding. Conduct cross-reactivity assessment through Western blot analysis across multiple species if using the antibody in comparative studies, verifying predicted reactivity percentages (e.g., 100% identity in human, dog, and guinea pig; 92% in rat) . Finally, employ orthogonal validation by comparing results using antibodies targeting different KHDRBS2 epitopes, as several commercial options target distinct regions from amino acids 30-79 to the C-terminus .

What are the key considerations for optimizing Western blot protocols when using KHDRBS2 antibodies?

Optimizing Western blot protocols for KHDRBS2 detection requires careful attention to several technical parameters. Start with appropriate protein extraction methods—KHDRBS2 localizes in both the nucleus and cytoplasm, so extraction buffers should effectively solubilize proteins from both cellular compartments . When loading samples, consider the predicted molecular weight of KHDRBS2 (approximately 39 kDa) to select appropriate gel percentage and molecular weight markers . For primary antibody incubation, follow recommended dilutions (typically 1:500-1:1000 for Western blotting with polyclonal KHDRBS2 antibodies) and optimize incubation conditions (temperature, duration, blocking solution composition) . Consider membrane stripping and reprobing requirements if detecting phosphorylated forms or performing co-immunoprecipitation studies. Implement appropriate controls including positive controls from validated cell lines (HeLa, 293T, Jurkat, K-562) , negative controls (KHDRBS2-knockdown samples), and loading controls to normalize expression levels. Finally, verify signal specificity by confirming the observed molecular weight matches the expected 39 kDa for KHDRBS2 .

How can researchers effectively study KHDRBS2's role in alternative splicing regulation using antibody-based approaches?

Studying KHDRBS2's role in alternative splicing requires sophisticated antibody-based methodologies that capture its dynamic interactions with RNA and splicing machinery. Implement RNA immunoprecipitation (RIP) assays using validated KHDRBS2 antibodies to isolate and identify KHDRBS2-bound RNA transcripts that may undergo alternative splicing regulation . Combine this with crosslinking immunoprecipitation (CLIP) techniques to identify direct RNA-binding sites with nucleotide precision. Employ co-immunoprecipitation (Co-IP) studies to identify KHDRBS2 protein interaction partners involved in splicing regulation, as demonstrated in studies examining KHDRBS2's interactions with other regulatory proteins . Utilize chromatin immunoprecipitation (ChIP) assays to investigate KHDRBS2's association with chromatin and potential co-transcriptional regulation of splicing, particularly at the promoter regions of target genes . Implement immunofluorescence microscopy to visualize KHDRBS2 localization in nuclear speckles or other subcellular compartments associated with splicing, noting that KHDRBS2 can localize in both the nucleus and cytoplasm . Finally, correlate antibody-based findings with functional splicing assays using minigene constructs to validate KHDRBS2's direct impact on alternative exon inclusion or exclusion in target transcripts.

What methodologies are recommended for investigating KHDRBS2's functional interactions with epigenetic regulators like KMT2D/WDR5?

Investigating KHDRBS2's interactions with epigenetic regulators requires integrating multiple antibody-dependent techniques. Begin with sequential Co-IP experiments using antibodies against KHDRBS2, KMT2D, and WDR5 to confirm protein-protein interactions and determine if these associations are direct or part of larger complexes . Implement proximity ligation assays (PLA) to visualize and quantify endogenous interactions between KHDRBS2 and epigenetic regulators in situ with subcellular resolution. Conduct ChIP-sequencing experiments to map genome-wide binding profiles of KHDRBS2, KMT2D, and WDR5, identifying regions of co-occupancy such as the KHDRBS2 promoter region where H3K4me3 accumulation occurs 1000-1500 bp upstream of the transcription start site . Perform ChIP-reChIP (sequential ChIP) to verify co-occupancy of KHDRBS2 with KMT2D/WDR5 at specific genomic loci. Utilize CRISPR-Cas9-mediated genomic editing to disrupt interaction domains followed by immunoprecipitation studies to determine the functional consequences of these interactions. Correlate antibody-based findings with histone modification analyses, particularly H3K4me3 levels at KHDRBS2 target genes, using ChIP followed by qPCR or Western blotting .

What experimental design would best elucidate KHDRBS2's role in the ACTBP2/KHDRBS2/HEY2 axis regulating blood-brain barrier permeability?

A comprehensive experimental design to investigate KHDRBS2's role in blood-brain barrier (BBB) regulation would integrate multiple approaches. First, establish appropriate cellular models using brain microvascular endothelial cells exposed to Aβ1-42 to replicate Alzheimer's disease-like conditions, where KHDRBS2 expression is significantly increased . Implement transendothelial electrical resistance (TEER) measurements and horseradish peroxidase (HRP) flux assays following KHDRBS2 knockdown or overexpression to directly quantify BBB permeability changes . Use immunofluorescence staining with KHDRBS2 antibodies combined with markers for tight junction proteins (ZO-1, occludin, claudin-5) to visualize and quantify KHDRBS2's impact on BBB integrity . Perform RNA-protein interaction studies including RNA immunoprecipitation to identify HEY2 mRNA binding to KHDRBS2 and assess how this affects mRNA stability. Conduct promoter activity assays examining how ACTBP2-mediated regulation of KHDRBS2 affects downstream target genes involved in BBB maintenance. Finally, validate in vitro findings using in vivo models such as APP/PS1 mice with KHDRBS2 knockdown, assessing BBB permeability through techniques such as Evans blue extravasation and immunohistochemical analysis of brain microvessel tight junction proteins .

How should researchers address inconsistencies between different KHDRBS2 antibodies when analyzing protein expression in tissue samples?

When facing inconsistent results with different KHDRBS2 antibodies, researchers should implement a systematic troubleshooting strategy. Begin with epitope mapping analysis to determine if discrepancies stem from antibodies recognizing different protein regions—KHDRBS2 antibodies target diverse epitopes ranging from amino acids 30-79 to the C-terminus, which may be differentially accessible in various tissue contexts . Perform isoform-specific analysis, as KHDRBS2 may have splice variants or post-translationally modified forms with altered epitope accessibility or expression patterns across tissues. Implement tissue-specific optimization by adjusting fixation methods, antigen retrieval protocols, and antibody dilutions for each antibody-tissue combination. Consider cross-reactivity assessment, particularly in tissues with expression of related family members like KHDRBS1 (Sam68) or KHDRBS3 that share structural similarities with KHDRBS2 . Validate observations using orthogonal methods such as RNA analysis (RT-qPCR, RNA-seq) to corroborate protein expression patterns. When publishing results, clearly document which antibody was used (including catalog number), validation methods performed, and experimental conditions to enable proper interpretation and reproducibility.

What strategies can resolve conflicting results between KHDRBS2 protein levels and functional outcomes in experimental models?

Resolving discrepancies between KHDRBS2 protein levels and functional outcomes requires multifaceted investigation of regulatory mechanisms. Assess KHDRBS2 subcellular localization using fractionation and immunofluorescence approaches, as functional activity may depend on nuclear versus cytoplasmic distribution rather than total protein levels . Examine post-translational modifications through immunoprecipitation followed by mass spectrometry or phospho-specific antibodies, as PTMs can drastically alter KHDRBS2 activity independent of expression levels. Investigate protein-protein interactions using co-immunoprecipitation studies to identify context-dependent binding partners that may enhance or inhibit KHDRBS2 function, as observed with KMT2D/WDR5 complexes . Evaluate RNA-binding activity through RNA immunoprecipitation assays, as KHDRBS2's primary function involves RNA interactions that may be regulated independently of protein abundance. Employ dose-response studies with graded KHDRBS2 expression to determine threshold effects and potential non-linear relationships between protein levels and functional outcomes. Finally, conduct temporal analyses to account for time-dependent changes in KHDRBS2 activity relative to protein expression, particularly in dynamic processes like BBB permeability regulation in response to Aβ1-42 exposure .

How should researchers interpret KHDRBS2 immunostaining patterns that show both nuclear and cytoplasmic localization?

Dual nuclear and cytoplasmic KHDRBS2 localization, as observed in immunofluorescence studies , requires careful interpretation considering KHDRBS2's multifunctional nature. First, validate the observed pattern using multiple antibodies targeting different KHDRBS2 epitopes to confirm the distribution is not antibody-specific. Perform subcellular fractionation followed by Western blotting to quantitatively confirm the distribution observed by immunostaining. Investigate condition-dependent shuttling by examining KHDRBS2 localization under various experimental conditions (e.g., serum starvation, cell cycle phases, Aβ1-42 treatment) to determine if the nuclear/cytoplasmic ratio changes in response to cellular stimuli . Consider isoform-specific localization patterns, as alternative splicing or post-translational modifications might direct different KHDRBS2 forms to distinct compartments. Correlate localization patterns with functional readouts such as RNA-binding activity, alternative splicing events, or BBB permeability to establish compartment-specific functions . Finally, employ super-resolution microscopy and co-localization studies with markers for specific nuclear (splicing speckles, chromatin regions) and cytoplasmic (RNA granules, stress granules) structures to precisely define the subnuclear and subcytoplasmic distribution of KHDRBS2.

How does KHDRBS2 function differ from other KHDRBS family members in the context of RNA processing and disease pathogenesis?

KHDRBS2 (also known as SLM1) shares structural features with other KHDRBS family members but exhibits distinct functional characteristics relevant to disease processes. Unlike the ubiquitously expressed KHDRBS1 (Sam68), KHDRBS2 shows more restricted tissue expression patterns, particularly in brain tissues, suggesting specialized neurological functions . KHDRBS2 contains a KH domain for RNA binding and signal transduction, but differs in its specific RNA target recognition sequences and downstream effector pathways compared to other family members . While all KHDRBS proteins regulate alternative splicing, KHDRBS2 appears to have a unique role in modulating blood-brain barrier permeability through the ACTBP2/KHDRBS2/HEY2 regulatory axis, particularly in the context of Alzheimer's disease pathology and Aβ1-42 microenvironments . KHDRBS2 distinctively influences tight junction protein expression (ZO-1, occludin, claudin-5) in brain microvascular endothelial cells, with knockdown studies demonstrating its direct impact on barrier integrity . KHDRBS2 is also uniquely regulated at the epigenetic level through interactions with KMT2D/WDR5 complexes that modify H3K4me3 marks in its promoter region, creating a specific regulatory mechanism not characterized for other family members .

What is the current evidence for KHDRBS2's involvement in neurodegenerative disorders, and how can antibody-based approaches advance this research?

Research indicates KHDRBS2 plays a significant role in neurodegenerative processes, particularly in Alzheimer's disease (AD) pathophysiology. Studies have demonstrated that KHDRBS2 expression is significantly increased in endothelial cells exposed to Aβ1-42, a hallmark peptide in AD pathology . KHDRBS2 functions within the ACTBP2/KHDRBS2/HEY2 regulatory axis to reduce expression of critical tight junction proteins (ZO-1, occludin, claudin-5), thereby increasing blood-brain barrier permeability in Aβ1-42 microenvironments . Knockdown experiments in APP/PS1 mouse models confirmed that reducing Khdrbs2 expression increases tight junction protein levels in brain microvessels, suggesting therapeutic potential . Antibody-based approaches can significantly advance this research through several strategies: using validated KHDRBS2 antibodies for immunohistochemical analysis of post-mortem human AD brain samples to correlate expression with disease severity; employing proximity ligation assays to investigate KHDRBS2's protein-protein interactions in situ in disease models; developing phospho-specific antibodies to study KHDRBS2 activation states in neurodegenerative contexts; utilizing ChIP-seq approaches to map genome-wide KHDRBS2 binding patterns in normal versus diseased brain tissues; and implementing single-cell immunofluorescence analyses to examine cell-type-specific KHDRBS2 expression in complex neural tissues.

What methodological considerations are essential when using KHDRBS2 antibodies to investigate its role in cancer progression and metastasis?

Investigating KHDRBS2's role in cancer requires specialized methodological considerations when using antibody-based approaches. Begin with comprehensive expression profiling across diverse tumor types and matched normal tissues using immunohistochemistry with validated KHDRBS2 antibodies, noting that KHDRBS2 has been implicated in cell proliferation, apoptosis, and tumorigenesis pathways . Implement tissue microarray analysis to correlate KHDRBS2 expression with clinicopathological parameters and patient outcomes while ensuring appropriate controls and consistent staining protocols. Consider potential heterogeneity by analyzing KHDRBS2 expression in different tumor regions, particularly at invasive fronts and metastatic sites. Examine subcellular localization patterns, as KHDRBS2's distribution between nuclear and cytoplasmic compartments may provide insights into its activity in different cancer contexts . Investigate epigenetic regulation of KHDRBS2 in cancer cells through ChIP assays examining histone modifications at its promoter, particularly the H3K4me3 marks regulated by KMT2D/WDR5 complexes . Assess KHDRBS2's impact on alternative splicing in cancer-relevant genes using RNA immunoprecipitation followed by sequencing (RIP-seq) or CLIP-seq. Finally, conduct functional validation through KHDRBS2 knockdown or overexpression in cancer cell lines and animal models, followed by comprehensive phenotypic analysis including proliferation, migration, invasion, and in vivo tumor growth assays.

What are the key differences between available KHDRBS2 antibodies that impact experimental applications?

The currently available KHDRBS2 antibodies exhibit several critical differences that significantly impact their experimental utility. Epitope recognition varies substantially: ABIN6745505 targets amino acids 30-79 , CAB6102 recognizes a sequence within amino acids 200 to the C-terminus , and other antibodies target regions like amino acids 160-349 or specifically the C-terminus . These differences affect accessibility in various experimental contexts, particularly for detecting protein interactions or post-translational modifications. Species reactivity profiles differ considerably, with some antibodies like ABIN6745505 demonstrating broad cross-reactivity across human, mouse, cow, dog, guinea pig, horse, monkey, and pig samples , while others have more restricted reactivity to human and mouse . This impacts comparative studies across model organisms. Application versatility varies, with certain antibodies validated only for Western blotting, while others are confirmed for multiple applications including Western blotting, immunohistochemistry-paraffin, ELISA, and flow cytometry . The table below provides a comparative analysis of key KHDRBS2 antibodies based on available specifications:

How can researchers optimize immunohistochemistry protocols for KHDRBS2 detection in different tissue samples?

Optimizing immunohistochemistry (IHC) protocols for KHDRBS2 detection requires methodical adjustment of multiple parameters based on tissue type and fixation method. Begin with fixation optimization—KHDRBS2 detection may be sensitive to fixation conditions, so compare paraformaldehyde, formalin, and alcohol-based fixatives to determine optimal epitope preservation. For antigen retrieval, systematically evaluate heat-induced epitope retrieval methods using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0) at varying temperatures and durations, as KHDRBS2 epitope accessibility may differ based on tissue type and fixation. When blocking, use tissue-specific optimization of blocking reagents (BSA, normal serum, commercial blockers) and durations to minimize background while preserving specific staining. For antibody parameters, carefully titrate primary antibody concentrations starting with recommended dilutions (1:100-1:200 for IHC-P with CAB6102) and optimize incubation conditions (temperature, duration). In signal development, compare sensitivity and specificity of different detection systems (ABC, polymer-based) and chromogens (DAB, AEC) for optimal signal-to-noise ratio. Implement comprehensive controls including positive controls (tissues known to express KHDRBS2), negative controls (primary antibody omission, isotype controls), and knockdown validation controls when possible. Document all optimization steps meticulously to ensure reproducibility across experiments and tissue types.

What techniques can effectively distinguish between KHDRBS2 and its closely related family members in experimental samples?

Distinguishing KHDRBS2 from related family members like KHDRBS1 (Sam68) and KHDRBS3 requires specialized techniques that maximize specificity. First, implement epitope-specific antibody selection by choosing KHDRBS2 antibodies targeting regions with minimal sequence homology to other family members, particularly avoiding the conserved KH domain . Conduct sequential immunoprecipitation where samples are first depleted of one family member before immunoprecipitating KHDRBS2 to reduce cross-reactivity. Employ isoform-specific PCR primers targeting unique regions of KHDRBS2 mRNA to correlate protein detection with transcript levels. Implement siRNA/shRNA validation with family member-specific knockdowns to confirm antibody specificity, as demonstrated in studies examining KHDRBS2 function . Utilize recombinant protein controls by performing Western blots with purified recombinant KHDRBS1, KHDRBS2, and KHDRBS3 proteins in parallel with experimental samples to establish specificity profiles for each antibody. Consider mass spectrometry validation of immunoprecipitated proteins to definitively identify KHDRBS2 versus related family members. Finally, employ cellular models with differential expression of family members (such as cell lines naturally expressing different ratios of KHDRBS proteins) to further validate antibody specificity in complex biological systems.