ABCC13 Antibody

What is ABCC13 and why is it significant in research despite being a pseudogene?

ABCC13 (ATP binding cassette subfamily C member 13) belongs to the superfamily of genes encoding ATP-binding cassette (ABC) transporters, which are responsible for transporting various molecules across cellular membranes. Although the human ABCC13 locus is considered a pseudogene incapable of encoding a functional ABC protein, it remains significant for research because it is part of the MRP subfamily involved in multi-drug resistance mechanisms . Despite lacking key functional motifs typically found in ABC proteins, ABCC13's expression patterns and alternative splicing suggest it may still play regulatory roles . The gene produces five distinct isoforms through alternative splicing, potentially contributing to diverse tissue-specific functions that warrant investigation even without transport activity .

What are the key considerations when selecting an ABCC13 antibody for research applications?

When selecting an ABCC13 antibody, researchers should consider several critical factors. First, determine the specific application requirements (WB, IHC, IF, ELISA, or IP) as different antibodies demonstrate variable efficacy across techniques . Second, verify species reactivity; most available ABCC13 antibodies react with human, mouse, and rat samples, but cross-reactivity should be confirmed for your specific model organism . Third, consider the antibody type—polyclonal antibodies often provide higher sensitivity but potentially lower specificity compared to monoclonals like the mouse IgM B-2 antibody . Fourth, evaluate the specific epitope recognition; some antibodies target specific amino acid regions (e.g., 56-105 aa) which may be absent in certain splice variants . Finally, confirm that validation data exists for your intended application, as demonstrated by western blot or IHC images showing specific detection patterns in relevant tissues .

What are the optimal protocols for detecting ABCC13 in western blot applications?

Successfully detecting ABCC13 via western blot requires careful optimization due to its pseudogene status and variable expression. Begin with sample preparation using RIPA buffer supplemented with protease inhibitors, followed by protein quantification to ensure equal loading (20-40 μg total protein per lane) . For SDS-PAGE, use 10-12% gels as ABCC13 has a molecular weight of approximately 31 kDa . After transfer to PVDF or nitrocellulose membranes, block with 5% BSA in TBST rather than milk, as milk proteins can sometimes interact with ABC family transporters . For primary antibody incubation, use ABCC13 antibodies at dilutions between 1:500-1:2000, incubating overnight at 4°C to maximize specific binding . Washing thoroughly (at least 3×10 minutes) with TBST helps reduce background signals. For detection, HRP-conjugated secondary antibodies against the appropriate host species (rabbit for polyclonal or mouse for monoclonal antibodies like B-2) should be used at 1:5000-1:10000 dilutions . Validated positive controls include extracts from 293 cells, COLO cells, and HT-29 cells, which have demonstrated reliable ABCC13 detection in previous studies .

How can researchers validate ABCC13 antibody specificity to ensure reliable immunohistochemistry results?

Validating ABCC13 antibody specificity for immunohistochemistry requires a multi-faceted approach. First, conduct parallel experiments with at least two different ABCC13 antibodies recognizing distinct epitopes to confirm consistent staining patterns . Second, perform peptide competition assays using the immunizing peptide (such as the 56-105 aa region) to demonstrate signal reduction or elimination . Third, include both positive controls (human colon tissue, where ABCC13 is highly expressed) and negative controls (tissues known to lack ABCC13 expression or using secondary antibody alone) . Fourth, compare protein detection with mRNA expression using techniques like RNA in situ hybridization or RT-PCR in the same tissue sections . Fifth, if available, use tissues from ABCC13 knockout models or cells treated with ABCC13-targeted siRNA as definitive negative controls . For paraffin-embedded sections, optimize antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0) and use antibody dilutions between 1:100-1:300 as recommended for most ABCC13 antibodies . Document all validation steps methodically, as this data substantiates the reliability of subsequent experimental findings.

What considerations are important when designing experiments to investigate potential functional roles of ABCC13 despite its pseudogene status?

Investigating ABCC13's functional roles requires specialized approaches given its pseudogene status. First, employ overexpression systems using tagged ABCC13 constructs (both full-length and individual splice variants) to identify potential protein-protein interactions through co-immunoprecipitation with validated antibodies . Second, utilize transcriptomics to examine correlations between ABCC13 expression levels and expression patterns of functional ABC transporters, which might reveal regulatory relationships . Third, conduct knockdown studies with siRNA or CRISPR-Cas9 targeting ABCC13 transcripts, followed by phenotypic assays and expression analysis of related genes to identify regulatory networks . Fourth, perform subcellular localization studies using immunofluorescence with antibodies like the rabbit polyclonal STJ91413 or mouse monoclonal B-2 to determine where ABCC13 protein products localize, potentially providing clues about function . Fifth, examine ABCC13 expression in disease models, particularly those involving drug resistance, as its MRP subfamily association suggests possible roles in this process despite lacking transport function . Finally, investigate whether ABCC13 transcripts function as competing endogenous RNAs (ceRNAs) that regulate other genes post-transcriptionally, a mechanism increasingly recognized for pseudogenes .

How should researchers address non-specific binding when using ABCC13 antibodies in western blot applications?

Non-specific binding is a common challenge when working with ABCC13 antibodies due to the pseudogene nature of the target and potential cross-reactivity with functional ABC transporters. To address this issue, implement a systematic troubleshooting approach. First, titrate primary antibody concentrations starting with higher dilutions (1:2000) and gradually increase concentration if needed, as excessive antibody often contributes to non-specific binding . Second, increase blocking stringency by extending blocking time to 2 hours and using 5% BSA with 0.5% Tween-20 in TBS . Third, perform more rigorous washing steps (5×10 minutes in TBST with gentle agitation) between antibody incubations . Fourth, pre-adsorb the antibody with non-relevant tissue lysates to remove cross-reactive antibodies before application to your experimental samples . Fifth, incorporate gradient gels (8-15%) to better separate proteins in the size range of interest, improving resolution around the expected 31 kDa ABCC13 band . If persistent bands appear at unexpected molecular weights, verify whether these might represent alternative splice variants, as ABCC13 is known to produce five different isoforms . Finally, consider using alternative detection methods such as fluorescent secondary antibodies which often provide cleaner backgrounds than chemiluminescence for challenging targets .

What approaches can resolve conflicting immunohistochemistry patterns when using different ABCC13 antibodies?

Conflicting immunohistochemistry patterns with different ABCC13 antibodies require systematic investigation to resolve. First, document the exact epitopes recognized by each antibody—for instance, whether they target different regions such as the 56-105 amino acid range versus other domains—as these may be differentially present in tissue-specific splice variants . Second, perform side-by-side comparisons using standardized protocols on serial tissue sections to directly compare staining patterns under identical conditions . Third, validate each antibody's specificity using peptide competition assays with their respective immunogens . Fourth, employ dual immunofluorescence staining with differently-conjugated secondary antibodies to simultaneously visualize both antibodies' binding patterns in the same section, revealing areas of overlap versus divergence . Fifth, complement protein detection with mRNA analysis using splice variant-specific probes to determine if discrepancies reflect genuine biological variation rather than technical artifacts . Sixth, consider cross-validation with orthogonal techniques such as in situ hybridization or RNAscope to confirm expression patterns at the transcript level . Finally, if discrepancies persist, prioritize data from antibodies with more extensive validation records and those that consistently align with transcript expression data from independent sources .

How can researchers differentiate between specific ABCC13 detection and potential cross-reactivity with other ABC transporter family members?

Differentiating specific ABCC13 detection from cross-reactivity with related ABC transporters requires rigorous controls and validation strategies. First, perform comparative sequence analysis of the antibody's immunizing peptide against other ABC transporters to identify potential cross-reactive family members based on epitope similarity . Second, include positive controls expressing known levels of ABCC13 alongside negative controls expressing related transporters but not ABCC13 . Third, conduct parallel detection experiments using gene-specific approaches like RT-PCR with primers targeting unique regions of ABCC13 to correlate protein detection with transcript presence . Fourth, perform knockdown validation using ABCC13-specific siRNAs and monitor the effect on antibody signal—true ABCC13 detection should diminish proportionally with successful knockdown while cross-reactive signals would remain unaffected . Fifth, utilize tissues or cells from model organisms where ABCC13 has been genetically deleted as definitive negative controls . Sixth, implement epitope mapping studies using peptide arrays to precisely identify the binding sites of antibodies showing suspected cross-reactivity . Finally, for critical experiments, consider using orthogonal identification methods such as mass spectrometry following immunoprecipitation to definitively identify the proteins being detected by your antibody of interest .

What are the emerging applications of ABCC13 antibodies in cancer research despite ABCC13's pseudogene status?

ABCC13 antibodies are finding novel applications in cancer research that extend beyond conventional functional studies. First, researchers are using ABCC13 antibodies to investigate pseudogene expression patterns across cancer types, with immunohistochemistry revealing unexpected ABCC13 expression in human liver carcinoma and other malignancies . Second, the correlation between ABCC13 expression and treatment response is being explored, as members of the MRP subfamily are implicated in multi-drug resistance, and ABCC13 may serve as a biomarker even without direct transport function . Third, ABCC13 antibodies are enabling studies of potential regulatory roles, where the pseudogene may act as a competing endogenous RNA (ceRNA) affecting post-transcriptional regulation of other ABC transporters . Fourth, investigations using co-immunoprecipitation with ABCC13 antibodies are revealing protein-protein interactions that suggest scaffold or regulatory functions in cancer cells . Fifth, researchers are examining splice variant expression using epitope-specific antibodies like the 56-105 aa targeting polyclonal antibody to determine if certain variants correlate with disease progression or treatment outcomes . Finally, dual staining approaches combining ABCC13 antibodies with markers of cancer stem cells are exploring whether ABCC13 expression identifies specific cellular subpopulations with clinical significance .

How can researchers design effective co-immunoprecipitation experiments using ABCC13 antibodies to identify novel protein interactions?

Designing effective co-immunoprecipitation (co-IP) experiments with ABCC13 antibodies requires careful consideration of several factors. First, select antibodies specifically validated for immunoprecipitation applications, such as the mouse monoclonal ABCC13 antibody (B-2), which has demonstrated efficacy in IP procedures . Second, optimize cell lysis conditions using gentle non-ionic detergents (0.5% NP-40 or 1% Triton X-100) to preserve protein-protein interactions while effectively solubilizing membrane-associated complexes where ABCC13 might reside . Third, pre-clear lysates thoroughly with protein A/G beads to reduce non-specific binding before adding the ABCC13 antibody . Fourth, determine optimal antibody amounts empirically (typically 2-5 μg per mg of protein lysate) and include isotype-matched control antibodies in parallel reactions to identify non-specific precipitants . Fifth, extend incubation times (overnight at 4°C with gentle rotation) to enhance the capture of weak or transient interactions . Sixth, implement stringent but appropriate washing conditions (typically 4-5 washes with decreasing salt concentrations) to remove non-specific binders while preserving genuine interactions . Finally, validate potential interacting partners using reverse co-IP and orthogonal methods such as proximity ligation assays or FRET to confirm the biological relevance of identified interactions . This comprehensive approach maximizes the likelihood of discovering meaningful ABCC13 protein interactions that could elucidate its non-canonical functions.

What potential roles might ABCC13 play in extracellular vesicle biology and how can researchers investigate this using available antibodies?

Investigating ABCC13's potential roles in extracellular vesicle (EV) biology represents an emerging research frontier that can be approached using available antibodies. First, researchers can employ differential ultracentrifugation to isolate EVs from cell culture supernatants or biological fluids, followed by western blotting with ABCC13 antibodies to determine if this pseudogene product is packaged into vesicles . Second, immunofluorescence co-localization studies using ABCC13 antibodies alongside established EV markers (CD63, CD9, TSG101) can reveal whether ABCC13 associates with multivesicular bodies or other EV biogenesis compartments . Third, immuno-electron microscopy using gold-conjugated secondary antibodies against primary ABCC13 antibodies can provide nanoscale resolution of ABCC13 localization on or within EVs . Fourth, researchers can perform proteomic analysis of EVs isolated from cells with ABCC13 knockdown versus controls to identify cargo differences that might indicate regulatory functions . Fifth, fluorescently labeled ABCC13 antibodies can be used for flow cytometry analysis of larger EVs to quantify the proportion carrying ABCC13 and correlate this with cellular origin or pathological states . Sixth, proximity labeling techniques combining ABCC13 antibodies with biotin ligase-conjugated secondary antibodies can identify proteins in close association with ABCC13 specifically in the EV compartment . These multifaceted approaches can help determine whether ABCC13, despite being a pseudogene product, might influence EV biogenesis, cargo selection, or recipient cell targeting through protein-protein interactions or regulatory mechanisms .

How can researchers integrate ABCC13 antibody-based findings with emerging genomic and transcriptomic data?

Integrating ABCC13 antibody-based findings with genomic and transcriptomic data requires a multi-dimensional approach. First, researchers should correlate protein expression patterns detected by antibodies with mRNA expression data from RNA-seq or microarray studies across matched tissues and cell types, identifying consistencies or discrepancies that might indicate post-transcriptional regulation . Second, analyze ABCC13 pseudogene expression in single-cell RNA-seq datasets to identify cell-type-specific expression patterns that can guide more precise immunohistochemistry studies with available antibodies . Third, overlay ABCC13 protein expression data with epigenetic profiles (DNA methylation, histone modifications) to understand regulatory mechanisms controlling its expression despite pseudogene status . Fourth, examine correlations between ABCC13 protein levels detected by antibodies and expression patterns of other genes within the ABC transporter family to identify potential compensatory or regulatory relationships . Fifth, integrate ABCC13 antibody-derived protein interaction data with protein-protein interaction networks from high-throughput studies to position ABCC13 within broader cellular signaling frameworks . Finally, correlate structural variations or mutations in the ABCC13 locus from whole-genome sequencing with alterations in protein expression or localization as detected by antibodies, potentially revealing structure-function relationships even for this non-canonical gene product . This integrative approach maximizes the value of antibody-based research by contextualizing findings within the broader molecular landscape.

What are the most promising future research directions for ABCC13 antibody applications in understanding disease mechanisms?

Future ABCC13 antibody research holds significant promise for understanding disease mechanisms across several domains. First, developing therapeutic applications through antibody-drug conjugates targeting ABCC13-expressing cancer cells represents an innovative approach, particularly given its differential expression in malignancies such as liver carcinoma . Second, exploring ABCC13's potential role in modulating immune responses through its expression in specialized immune cell populations could open new avenues in immunotherapy research, detectable through flow cytometry with fluorescently-labeled ABCC13 antibodies . Third, investigating ABCC13's involvement in neurodegenerative disorders through detailed immunohistochemical mapping of its expression in brain regions affected by conditions like Alzheimer's or Parkinson's disease might reveal unexpected disease associations . Fourth, examining ABCC13's potential contribution to drug resistance mechanisms in cancer through correlative studies of its expression patterns with treatment responses could identify its utility as a prognostic biomarker . Fifth, exploring ABCC13's role in developmental biology using antibodies to track its expression throughout embryogenesis and organogenesis might uncover previously unrecognized functions . Finally, investigating potential non-canonical functions of ABCC13 through comprehensive interactome mapping using co-immunoprecipitation with highly specific antibodies could reveal unexpected signaling or regulatory roles that contribute to disease pathogenesis . These research directions collectively represent promising avenues for leveraging ABCC13 antibodies to advance our understanding of diverse disease mechanisms.