acbd5b Antibody

What is ACBD5 and why is it significant for research?

ACBD5, also known as RDLKD, is a member of the acyl-Coenzyme A binding protein family that functions primarily in the transport and distribution of long chain acyl-Coenzyme A in cells. It is located in the peroxisome membrane and is ubiquitinated in brain and kidney tissue. The protein may play a significant role in the differentiation of megakaryocytes and formation of platelets. ACBD5's involvement in peroxisome-ER membrane contacts makes it particularly interesting for researchers studying organelle interactions and lipid metabolism disorders. Recent studies have highlighted its importance in very long-chain fatty acid (VLCFA) metabolism, with ACBD5 deficiency linked to abnormal VLCFA accumulation .

What are the typical applications for ACBD5 antibody in research?

ACBD5 antibody has several validated research applications, with Western Blot (WB) and Immunohistochemistry (IHC) being the most common. The antibody has been validated for ELISA applications as well. Published literature documents successful use in WB applications across multiple studies. The antibody shows confirmed reactivity with human and mouse samples, making it suitable for comparative studies across these species. For Western Blot applications, the recommended dilution range is 1:5000-1:50000, while IHC applications typically use dilutions between 1:500-1:2000 .

What is known about ACBD5's molecular characteristics?

ACBD5 is composed of 534 amino acids with a calculated molecular weight of 60 kDa, although it is typically observed at 60-70 kDa in experimental conditions. The gene has been assigned the NCBI Gene ID 91452 and UniProt ID Q5T8D3. The GenBank accession number for the reference sequence is BC030555. The protein contains an acyl-CoA binding domain that is essential for its function in fatty acid metabolism. Studies using mutated ACB domains have demonstrated that this region is required for proper metabolism of VLCFAs, as complementation experiments with ACBD5 containing mutated ACB domains failed to restore normal VLCFA metabolism in knockout cells .

What characteristics should researchers consider when selecting an ACBD5 antibody?

When selecting an ACBD5 antibody, researchers should consider several critical parameters: antibody type (monoclonal vs. polyclonal), host species, immunogen used for production, validated applications, and cross-reactivity profile. For example, the polyclonal antibody referenced in the literature (21080-1-AP) is rabbit-derived and was generated using an ACBD5 fusion protein (Ag15287) as immunogen. Researchers should select antibodies that have been validated for their specific application (WB, IHC, ELISA) and confirm reactivity with their species of interest. Additionally, consider the observed molecular weight (60-70 kDa for ACBD5) when verifying antibody specificity in your experimental system .

How do storage conditions affect ACBD5 antibody performance?

Proper storage of ACBD5 antibody is crucial for maintaining its reactivity and specificity. The referenced ACBD5 antibody should be stored at -20°C and remains stable for one year after shipment under these conditions. The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. According to manufacturer specifications, aliquoting is unnecessary for -20°C storage, which differs from many other antibodies that require aliquoting to prevent freeze-thaw cycles. For small 20μl preparations, the formulation may contain 0.1% BSA as a stabilizer. Researchers should always check expiration dates and follow manufacturer-specific storage recommendations, as variations exist between products .

What dilution ranges are optimal for different ACBD5 antibody applications?

Optimal dilution ranges for ACBD5 antibody vary significantly by application:

These ranges provide starting points, but researchers should perform titration experiments within these ranges to determine optimal conditions for their specific sample types and experimental systems. As noted in the technical information, the optimal dilution is sample-dependent and should be empirically determined for each experimental setup .

What protocols are recommended for ACBD5 detection in immunohistochemistry?

For immunohistochemical detection of ACBD5, antigen retrieval is a critical step that significantly impacts results. The recommended protocol suggests using TE buffer at pH 9.0 for antigen retrieval, although citrate buffer at pH 6.0 can serve as an alternative. Positive IHC detection has been successfully demonstrated in human ovary cancer tissue and human stomach tissue. For dilution, start with a range of 1:500-1:2000 and optimize based on your specific tissue and staining system. When performing IHC with ACBD5 antibody, include appropriate positive controls (such as human ovary cancer tissue or stomach tissue) where ACBD5 expression has been verified. Use serial dilutions to determine optimal antibody concentration for your specific tissue sample and detection system .

How should researchers design Western blot experiments for optimal ACBD5 detection?

For Western blot detection of ACBD5, researchers should consider several optimization factors. The antibody has been validated in multiple cell lines including HeLa cells, L02 cells, MCF-7 cells, and mouse skeletal muscle tissue, which can serve as positive controls. Start with a dilution in the recommended range (1:5000-1:50000) and adjust based on signal strength. Since ACBD5 has a calculated molecular weight of 60 kDa but is typically observed between 60-70 kDa, use appropriate molecular weight markers spanning this range. Optimization may include adjusting blocking conditions, incubation times, and detection methods. Consider using gradient gels (e.g., 4-12%) to better resolve proteins in this molecular weight range, and validate specificity using ACBD5 knockout or knockdown samples when available .

What approaches can be used to study ACBD5 function in peroxisome-ER contacts?

Studies of ACBD5's role in peroxisome-ER contacts employ several sophisticated approaches. Researchers have used CRISPR/Cas9 to generate ACBD5 knockout cell lines, followed by rescue experiments with wild-type or mutant ACBD5 constructs. The FlpIn system has been successfully employed to generate stable cell lines expressing ACBD5 or related proteins like ACBD4. Peroxisome-ER contacts can be visualized and quantified using fluorescence microscopy with appropriate organelle markers. For functional studies, researchers have implemented D3-C22:0 loading tests to assess VLCFA metabolism, measuring levels of labeled C26:0 and C16:0 to evaluate beta-oxidation efficiency. Complementation studies comparing wild-type ACBD5 with ACBD5 containing a mutated ACB domain have demonstrated the importance of this domain for proper VLCFA metabolism .

How can ACBD5 antibodies be used to investigate peroxisome-ER membrane contacts?

ACBD5 antibodies can be powerful tools for investigating peroxisome-ER membrane contacts through multiple advanced approaches. Researchers can employ immunofluorescence co-localization studies using ACBD5 antibodies alongside ER markers to visualize contact sites. Super-resolution microscopy techniques (STED, STORM, or PALM) can provide nanoscale resolution of these contacts. For biochemical validation, proximity ligation assays combining ACBD5 antibodies with antibodies against ER-resident proteins can confirm protein interactions at contact sites. Immunoprecipitation using ACBD5 antibodies followed by mass spectrometry can identify novel interaction partners at these junctions. Research has demonstrated that ACBD5 forms tethering complexes connecting peroxisomes and ER, and knockout studies show that loss of ACBD5 reduces these contacts substantially. Interestingly, overexpression of ACBD4 in ACBD5 knockout cells can restore peroxisome-ER contacts, suggesting functional redundancy between these proteins in tethering mechanisms .

What approaches can resolve the functional differences between ACBD4 and ACBD5?

Despite structural similarities, ACBD4 and ACBD5 exhibit distinct functional properties that require sophisticated experimental approaches to differentiate. Comparative knockout studies have revealed that while both proteins contribute to peroxisome-ER contacts, they have different impacts on VLCFA metabolism. ACBD5 knockout results in increased VLCFA levels and decreased beta-oxidation of C26:0, whereas ACBD4 knockout shows unaltered VLCFA levels despite slightly increased rates of VLCFA beta-oxidation. To further investigate these differences, researchers can employ domain-swapping experiments, creating chimeric proteins to identify regions responsible for functional specificity. Lipidomic analyses comparing knockout cell lines provide detailed insights into their differential roles in lipid metabolism. Complementation experiments using the FlpIn system to express ACBD4 in ACBD5 knockout cells have demonstrated that ACBD4 can restore peroxisome-ER contacts but may not fully complement all ACBD5 functions in VLCFA metabolism, highlighting their distinct roles despite overlapping capabilities .

What methodologies can determine if ACBD5 dysfunction contributes to disease phenotypes?

Investigating ACBD5's potential role in disease requires multilayered approaches. Patient-derived samples can be analyzed using ACBD5 antibodies to assess protein expression and localization alterations in disease states. For functional validation, CRISPR/Cas9-engineered cell lines mimicking patient mutations can be created and analyzed for peroxisome-ER contacts and VLCFA metabolism. D3-C22:0 loading tests measuring D3-C26:0 accumulation and D3-C16:0/D3-C26:0 ratios provide insights into VLCFA processing defects. Rescue experiments with wild-type ACBD5 expression in patient-derived cells can confirm if phenotypes are directly attributable to ACBD5 dysfunction. Research has shown that ACBD5 with a mutated ACB domain cannot complement VLCFA metabolism defects in knockout cells, suggesting that mutations affecting this domain might contribute to disease phenotypes characterized by VLCFA accumulation. These methodologies can be particularly relevant for investigating peroxisomal disorders where abnormal VLCFA metabolism is a hallmark feature .

What are common challenges in obtaining specific ACBD5 detection and how can they be overcome?

Researchers may encounter several challenges when detecting ACBD5, including non-specific binding, suboptimal signal intensity, and variable results across samples. To improve specificity in Western blots, optimize blocking conditions (consider 5% non-fat milk or BSA in TBST) and validate antibody specificity using ACBD5 knockout or knockdown samples as negative controls. For weak signals in IHC, antigen retrieval is critical—compare the recommended TE buffer (pH 9.0) with citrate buffer (pH 6.0) to determine optimal conditions for your tissue. If experiencing variability across experiments, standardize protein loading for Western blots and implement consistent tissue processing protocols for IHC. Background reduction in IHC can be achieved by titrating primary antibody concentration and optimizing incubation times and temperatures. For enhanced detection sensitivity in challenging samples, consider signal amplification methods such as tyramide signal amplification or polymer detection systems .

How can researchers optimize experiments to study ACBD5's role in VLCFA metabolism?

To effectively study ACBD5's role in VLCFA metabolism, researchers should consider several optimization strategies. D3-C22:0 loading tests have proven valuable for assessing VLCFA processing—optimize substrate concentration and incubation time for your specific cell type. For lipidomic analyses, sample preparation is critical; standardize cell numbers and growth conditions across experimental groups. When performing complementation studies, ensure comparable expression levels of wild-type and mutant ACBD5 constructs to facilitate accurate functional comparisons. Studies have shown that the D3-C16:0/D3-C26:0 ratio serves as a sensitive indicator of beta-oxidation efficiency—calculate this ratio consistently across experiments to detect subtle metabolic alterations. Research has demonstrated that ACBD5 knockout cells show increased levels of VLCFAs and decreased beta-oxidation of C26:0, while expression of wild-type ACBD5 (but not ACB domain mutants) can restore normal VLCFA metabolism, emphasizing the importance of the ACB domain for this function .

How does ACBD5 compare to other peroxisomal membrane proteins in research applications?

ACBD5 represents one of several peroxisomal membrane proteins studied in current research, but has distinctive features that differentiate it from others. Unlike PEX proteins that primarily function in peroxisome biogenesis, ACBD5 specializes in organelle contacts and lipid metabolism. When designing experiments, researchers should consider that ACBD5 contains both an acyl-CoA binding domain and a FFAT motif that enables ER interaction, a combination not found in most other peroxisomal proteins. This unique domain architecture facilitates its dual role in both lipid metabolism and inter-organelle communication. Experimental approaches that have proven successful for ACBD5, such as D3-C22:0 loading tests and peroxisome-ER contact quantification, may need modification when studying other peroxisomal membrane proteins with different functions. Research has demonstrated that while other proteins like ACBD4 can partially complement ACBD5 function in peroxisome-ER contacts, they may not fully restore all ACBD5-dependent processes, highlighting the unique aspects of ACBD5 biology compared to other peroxisomal proteins .

What methodological advances are enhancing our understanding of ACBD5 function?

Recent methodological advances have significantly expanded our understanding of ACBD5 biology. CRISPR/Cas9 gene editing has enabled precise creation of knockout cell lines, allowing detailed functional characterization. Advanced imaging techniques, including super-resolution microscopy, have improved visualization of peroxisome-ER contacts at nanoscale resolution. Sophisticated lipidomic approaches now provide comprehensive profiling of lipid changes in ACBD5-deficient cells, moving beyond targeted analysis of specific VLCFAs. Isotope-labeled fatty acid loading tests (such as D3-C22:0) offer dynamic assessment of VLCFA metabolism rather than static measurements. The FlpIn system for generating stable cell lines with controlled expression of wild-type or mutant ACBD5 has enabled precise complementation studies. These methodological advances collectively allow researchers to move from correlative to causative understanding of ACBD5 function in both normal physiology and disease states .

What are the most significant recent advances in ACBD5 research?

Recent ACBD5 research has yielded several significant advances. Studies have definitively established ACBD5's role in forming tethering complexes that physically connect peroxisomes and the ER, enhancing our understanding of organelle communication networks. Detailed functional characterization has revealed ACBD5's importance in VLCFA metabolism, with knockout studies demonstrating increased VLCFA levels and decreased beta-oxidation in its absence. The discovery that ACBD4 can partially complement ACBD5 function in peroxisome-ER contacts but may not fully restore all metabolic functions has highlighted both redundancy and specificity in these related proteins. Structure-function studies have identified the critical importance of the ACB domain for VLCFA metabolism, as ACBD5 with mutated ACB domains fails to complement metabolic defects in knockout cells. These advances collectively provide a more nuanced understanding of ACBD5's multifaceted roles in cellular physiology and establish a foundation for exploring its potential contributions to human disease .