CRABP2 Antibody

What is CRABP2 and why is it important in research?

CRABP2 is a 14 kDa cytosolic protein belonging to the fatty acid binding protein family. It functions by binding bioactive retinoic acid and transporting it to the nucleus, where it facilitates transfer to the retinoic acid receptor, unlike CRABP1 which has a different functional mechanism. Human CRABP2 consists of 138 amino acids with a nuclear localization signal (amino acids 21-31) and a series of beta-strands forming a beta-barrel that accommodates retinoic acid internally (amino acids 41-135). CRABP2 is expressed in multiple cell types including keratinocytes, corneal epithelium, fibroblasts, and visceral smooth muscle cells . The protein has gained significant research interest due to its involvement in cell proliferation, apoptosis, and metastasis through retinoic acid signaling pathways, with aberrant expression linked to various cancers including skin, lung, and liver malignancies .

What are the key considerations when selecting a CRABP2 antibody for my research?

When selecting a CRABP2 antibody, researchers should consider several critical factors: (1) Antibody specificity - verify the antibody has been validated against the specific species of interest, as CRABP2 antibodies show cross-reactivity across human, mouse, and rat tissues with specific band detection at approximately 14-16 kDa; (2) Application compatibility - confirm the antibody has been validated for your specific application (Western blot, IHC, or IF) as demonstrated in the literature; (3) Clonality - choose between polyclonal antibodies like AF5740 or monoclonal antibodies like EPR17376 based on your experimental needs; (4) Detection system compatibility - ensure the antibody works with your secondary detection system; and (5) Validated performance - review published literature demonstrating successful use in similar experimental conditions . Antibody dilution optimization is also essential, with Western blot protocols typically using 0.5-1.0 μg/mL for polyclonal antibodies or 1/1000 dilution for monoclonal antibodies .

How do CRABP2 expression patterns differ across tissue types?

CRABP2 exhibits distinct expression patterns across different tissue types, which is important to consider when designing experiments. Immunohistochemical analyses reveal that CRABP2 demonstrates both cytoplasmic and nuclear staining in stratified squamous epithelium and hair follicle cells in human, mouse, and rat skin tissues . In skin tissues specifically, CRABP2 is found in the stratified squamous epithelium, hair follicle cells, and sweat gland cells, with notable differences in intensity between species . In pathological tissues, CRABP2 displays cytoplasmic and nuclear staining in human pancreatic ductal adenocarcinoma . Additionally, CRABP2 shows expression in colorectal adenocarcinoma cell lines like HT-29, with both nuclear and cytoplasmic localization . Plasma levels of CRABP2 are significantly elevated in patients with early-stage lung adenocarcinoma (31.6587 ±13.8541 ng/mL) compared to healthy controls (13.9328 ± 5.5805 ng/mL) . These tissue-specific expression patterns make CRABP2 a valuable marker for both normal tissue characterization and cancer research.

What are the optimal protocols for Western blot detection of CRABP2?

For optimal Western blot detection of CRABP2, researchers should follow these methodological guidelines: First, prepare protein lysates from your samples of interest, such as cancer cell lines (MCF-7, MDA-MB-468, T47D) or tissue samples (skin, liver, lung) . For PVDF membrane transfers, use reducing conditions and appropriate immunoblot buffers such as Immunoblot Buffer Group 1 . For primary antibody incubation, human CRABP2 polyclonal antibodies typically work best at 0.5 μg/mL concentration, while monoclonal antibodies like EPR17376 work well at 1/1000 dilution . When selecting secondary antibodies, HRP-conjugated anti-goat IgG or anti-rabbit IgG (depending on your primary antibody host species) should be used at manufacturer-recommended dilutions (typically 1/100000 for Western blot) . The predicted band size for CRABP2 is 16 kDa, but the observed band typically appears at approximately 14 kDa . Visualization requires appropriate exposure times, with 3 minutes being sufficient in many documented protocols . For troubleshooting, verify protein loading with appropriate housekeeping controls and consider using breast cancer cell lines as positive controls, as they consistently express detectable levels of CRABP2 .

How should I optimize immunohistochemistry (IHC) protocols for CRABP2 detection in different tissue samples?

For optimized immunohistochemistry detection of CRABP2 across different tissue samples, implement the following methodological approach: Begin with heat-mediated antigen retrieval using Tris/EDTA buffer at pH 9.0, which is critical for exposing the CRABP2 epitope . For paraffin-embedded tissues (human skin, pancreatic ductal adenocarcinoma, mouse skin, or rat skin), a 1/1000 dilution of anti-CRABP2 antibody [EPR17376] has been validated to provide specific staining . Block non-specific binding using 5% non-fat dry milk in TBST before primary antibody incubation . For secondary detection, HRP-conjugated anti-rabbit IgG at 1/500 dilution followed by appropriate chromogenic substrate development provides strong signal with minimal background . Always include a secondary antibody-only control using PBS instead of primary antibody to assess non-specific binding . When evaluating results, expect to observe cytoplasmic and nuclear staining in specific cell types: stratified squamous epithelium and hair follicle cells in skin samples, and cancer cells in adenocarcinoma samples . For multi-species studies, be aware that CRABP2 antibodies can detect the protein in human, mouse, and rat tissues with similar staining patterns, making comparative studies feasible .

What are the best practices for immunofluorescence detection of CRABP2?

For optimal immunofluorescence detection of CRABP2, follow these established methodological steps: Begin by fixing cells with 4% paraformaldehyde to preserve protein localization and cellular architecture . Cell permeabilization should be performed using 0.2% Triton X-100 to allow antibody access to intracellular CRABP2 . Block non-specific binding with 5% BSA before primary antibody incubation . For primary antibody incubation, anti-CRABP2 antibodies have been successfully used at 1:50 dilution (Abnova antibodies) or 1:250 dilution (EPR17376) depending on the specific antibody . For visualization, use species-appropriate secondary antibodies conjugated with fluorophores such as Alexa Fluor 488 at 1:200 dilution . For co-localization studies, CRABP2 can be effectively paired with proliferation markers like Ki-67 (1:1,000 dilution) . Include DAPI nuclear counterstaining to facilitate subcellular localization analysis . When analyzing results, expect both nuclear and cytoplasmic staining in cells expressing CRABP2, such as HT-29 colorectal adenocarcinoma cells . For optimal imaging, use confocal microscopy to clearly distinguish between nuclear and cytoplasmic localization patterns . Always include appropriate negative controls and consider using cell lines with known CRABP2 expression as positive controls.

How can I address common issues with non-specific binding when using CRABP2 antibodies?

Non-specific binding is a common challenge when working with CRABP2 antibodies that can be systematically addressed through several methodological approaches. First, optimize blocking conditions by testing different blocking agents; 5% non-fat dry milk in TBST has proven effective for Western blot applications, while 5% BSA works well for immunofluorescence studies . Titrate antibody concentrations; for most applications, Western blot detection works optimally with 0.5 μg/mL of polyclonal antibodies or 1/1000 dilution of monoclonal antibodies, while immunohistochemistry may require 1/1000 dilution with monoclonal antibodies . Always include proper controls, particularly secondary antibody-only controls using PBS instead of primary antibody to identify non-specific secondary antibody binding . For tissue samples, perform adequate antigen retrieval using heat-mediated methods with Tris/EDTA buffer at pH 9.0 before antibody application . If background persists in Western blots, consider more stringent washing conditions or alternative buffer systems like Immunoblot Buffer Group 1 . For immunohistochemistry applications with continued background issues, try increasing the number and duration of wash steps between antibody incubations. Always validate antibody specificity by confirming the observed band matches the expected molecular weight (approximately 14 kDa for CRABP2) .

What strategies can I employ when CRABP2 detection signals are weak or inconsistent?

When encountering weak or inconsistent CRABP2 detection signals, implement these evidence-based troubleshooting strategies: First, verify sample integrity and protein concentration through appropriate protein quantification methods and use of housekeeping protein controls in Western blot applications. For Western blot applications specifically, increase protein loading (10-20 μg per lane has been validated for skin lysates) , optimize primary antibody concentration (try increasing from 0.5 μg/mL to 1.0 μg/mL for polyclonal antibodies) , extend primary antibody incubation time (overnight at 4°C often improves signal), and consider signal amplification systems or more sensitive detection reagents. For immunohistochemistry and immunofluorescence, enhance antigen retrieval by optimizing buffer composition, pH, and heating time/temperature using Tris/EDTA buffer pH 9.0 as a starting point . Extend antibody incubation times or increase antibody concentrations within validated ranges. Use signal amplification systems like tyramide signal amplification if appropriate for your detection system. Choose appropriate positive controls with known high CRABP2 expression such as MCF-7, MDA-MB-468, or T47D breast cancer cell lines , or HT-29 colorectal adenocarcinoma cells . Consider tissue or cell type-specific expression patterns, as CRABP2 shows variable expression across different tissues and may be upregulated in certain cancer types .

How can I validate the specificity of CRABP2 antibody detection in my experimental system?

To rigorously validate CRABP2 antibody specificity in your experimental system, implement a multi-faceted approach: First, perform molecular weight verification by confirming that your detected band corresponds to the expected molecular weight of CRABP2 (predicted at 16 kDa, but typically observed at approximately 14 kDa in reducing conditions) . Implement positive and negative controls by using cell lines or tissues with known CRABP2 expression patterns - breast cancer cell lines (MCF-7, MDA-MB-468, T47D) and skin tissues serve as reliable positive controls . Conduct RNA interference validation by knocking down CRABP2 using shRNA or siRNA technology and confirming corresponding reduction in antibody signal . For more rigorous validation, perform peptide competition assays where pre-incubation of the antibody with excess purified CRABP2 protein or immunizing peptide should abolish specific binding. Consider dual-detection methods by using two different antibodies targeting distinct epitopes of CRABP2 to confirm consistent detection patterns. In tissue samples, verify cellular and subcellular localization patterns match known CRABP2 distribution (both nuclear and cytoplasmic staining in expressing cells) . For comprehensive validation, correlate protein detection with mRNA expression data when available. Finally, consider cross-species reactivity validation if working across multiple species, as CRABP2 antibodies have demonstrated reactivity with human, mouse, and rat CRABP2 .

What mechanisms underlie CRABP2's role in tumorigenesis and how can they be experimentally investigated?

CRABP2's role in tumorigenesis involves several key cellular mechanisms that can be experimentally investigated through specific methodological approaches. The primary mechanism involves CRABP2's function as a retinoic acid (RA) transport protein that shuttles between cytoplasm and nucleus, delivering RA to nuclear receptors and acting as a coactivator of retinoic acid receptor (RAR), thereby regulating gene expression related to cell proliferation, apoptosis, and metastasis . To investigate this mechanism, researchers can employ RNA interference techniques (shRNA or siRNA) to knock down CRABP2 expression and observe phenotypic changes in cell proliferation, migration, invasion, and apoptosis using established assays like CCK-8, EdU staining, transwell assays, and flow cytometry . Molecular pathway analysis reveals that CRABP2 silencing results in decreased expression of ERK/VEGF pathway-related proteins while increasing apoptosis-related protein expression, suggesting these as key downstream effectors . For in vivo validation, xenograft models with CRABP2-silenced cancer cells demonstrate reduced tumor development, confirming its role in tumorigenesis . In specific cancers like lung adenocarcinoma, CRABP2 overexpression correlates with disease progression and poor prognosis, making it a potential biomarker and therapeutic target . Interestingly, in retinoic acid-resistant breast cancer, activation of the CRABP2/RAR pathway using compounds like curcumin can restore retinoic acid-mediated apoptosis, suggesting a complex role depending on cellular context .

How can CRABP2 antibodies be utilized in developing cancer biomarkers and therapeutic strategies?

CRABP2 antibodies offer significant potential for cancer biomarker development and therapeutic strategy design based on recent research findings. For biomarker applications, CRABP2 antibodies can be employed in developing ELISA or other immunoassay-based diagnostic tests for early cancer detection, particularly for lung adenocarcinoma where plasma CRABP2 levels show promising diagnostic accuracy (sensitivity 70.98%, specificity 94.53%, AUC 0.839) for early-stage disease . Immunohistochemical detection of CRABP2 in tissue biopsies can help with cancer classification and prognosis prediction, as CRABP2 expression correlates with tumor stage progression in several cancers . For monitoring treatment response, tracking CRABP2 levels before and after therapy may provide valuable information about treatment efficacy, particularly for therapies targeting retinoic acid pathways. In therapeutic applications, CRABP2 antibodies can be used for target validation in drug development pipelines focusing on retinoic acid signaling. They are essential tools for screening compounds that may modulate CRABP2 function or expression, like the demonstrated effect of curcumin on the CRABP2/RAR pathway in retinoic acid-resistant breast cancer . Additionally, CRABP2 antibodies enable mechanism-of-action studies for novel therapeutics targeting this pathway. For developing advanced immunotherapeutic approaches, antibody-drug conjugates targeting CRABP2-expressing cancer cells could potentially deliver cytotoxic payloads specifically to cancer cells while sparing normal tissues with lower CRABP2 expression.

How can I effectively use CRABP2 antibodies in multi-parameter flow cytometry for cancer research?

For effective multi-parameter flow cytometry utilizing CRABP2 antibodies in cancer research, implement these methodological guidelines: Begin with optimized cell preparation by creating single-cell suspensions from fresh tissue samples or cultured cells, followed by fixation with 4% paraformaldehyde to preserve cellular architecture . For intracellular proteins like CRABP2, permeabilization with 0.2% Triton X-100 or commercial permeabilization buffers is essential for antibody accessibility . Select compatible fluorophore-conjugated CRABP2 antibodies or use unconjugated primary antibodies followed by fluorophore-conjugated secondary antibodies. For multi-parameter panels, combine CRABP2 detection with relevant cancer markers (e.g., proliferation markers like Ki-67, which has been successfully co-stained with CRABP2) . When designing panels, carefully select fluorophores with minimal spectral overlap and implement proper compensation controls. Include appropriate controls: unstained cells, isotype controls, fluorescence-minus-one (FMO) controls, and positive controls from cell lines with known CRABP2 expression (such as breast cancer cell lines MCF-7, MDA-MB-468, or T47D) . For analysis, identify CRABP2-positive populations and correlate with other cancer-related markers to investigate associations with cell cycle status, stemness, or differentiation markers. This approach is particularly valuable for studying heterogeneity in CRABP2 expression within tumor populations and for identifying specific cell subpopulations with elevated CRABP2 expression that may correlate with aggressive phenotypes or treatment resistance.

What approaches are effective for studying CRABP2 protein-protein interactions in cancer signaling pathways?

For investigating CRABP2 protein-protein interactions in cancer signaling pathways, researchers should employ multiple complementary methodological approaches: Co-immunoprecipitation (Co-IP) using CRABP2 antibodies can pull down CRABP2 along with its interacting partners, followed by Western blot or mass spectrometry identification of binding partners. When performing Co-IP, use appropriate lysis buffers that preserve protein-protein interactions while effectively extracting CRABP2 from both cytoplasmic and nuclear compartments. Proximity ligation assay (PLA) can visualize and quantify CRABP2 interactions with suspected binding partners (particularly retinoic acid receptors) within intact cells with spatial resolution. For more comprehensive interaction mapping, BioID or APEX2 proximity labeling with CRABP2 fusion proteins can identify proteins in close proximity to CRABP2 in living cells. Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) can detect direct protein-protein interactions in living cells, particularly useful for studying dynamic interactions between CRABP2 and nuclear receptors during retinoic acid signaling. Functional validation of identified interactions can be performed through domain mapping and mutagenesis studies to identify specific regions of CRABP2 involved in various protein interactions. For pathway analysis, combine interaction data with signaling pathway inhibition studies targeting ERK/VEGF pathways, which have been implicated in CRABP2-mediated cancer progression . This multi-faceted approach can help elucidate how CRABP2 influences cancer cell behavior through its interactions with retinoic acid receptors and other signaling proteins.

How can advanced imaging techniques enhance our understanding of CRABP2 subcellular dynamics in cancer cells?

Advanced imaging techniques offer powerful approaches for elucidating CRABP2 subcellular dynamics in cancer cells with unprecedented spatial and temporal resolution. Live-cell imaging with fluorescently tagged CRABP2 can reveal real-time shuttling between cytoplasmic and nuclear compartments in response to retinoic acid stimulation or other cellular signals. For such applications, construct CRABP2-GFP fusion proteins and validate that they retain normal localization and function. Super-resolution microscopy techniques (STED, STORM, or PALM) can overcome the diffraction limit to visualize CRABP2 distribution with nanometer precision, potentially revealing previously undetectable localization patterns within nuclear subdomains or other cellular compartments. For high-resolution co-localization studies, employ multi-color super-resolution imaging to examine CRABP2's spatial relationship with retinoic acid receptors or components of the ERK/VEGF pathway identified in functional studies . Fluorescence recovery after photobleaching (FRAP) can measure CRABP2 mobility in different cellular compartments, providing insights into its binding dynamics with retinoic acid and nuclear receptors. For studying nuclear-cytoplasmic shuttling kinetics, use photoactivatable or photoconvertible CRABP2 fusion proteins to track specific protein populations over time. Correlative light and electron microscopy (CLEM) combines the specificity of fluorescence imaging with the ultrastructural context provided by electron microscopy, potentially revealing CRABP2 association with specific subcellular structures. These advanced imaging approaches can significantly enhance our understanding of how altered CRABP2 dynamics contribute to cancer pathogenesis through mechanisms such as dysregulated retinoic acid signaling and abnormal nuclear receptor interactions.

How might CRABP2 antibodies be utilized in studying treatment resistance mechanisms in cancer?

CRABP2 antibodies offer valuable tools for investigating treatment resistance mechanisms in cancer through several research approaches. In retinoic acid-resistant cancers, CRABP2 antibodies can help characterize alterations in CRABP2 expression and localization that may contribute to therapy resistance, building on findings that the CRABP2/RAR pathway can be reactivated by compounds like curcumin to induce apoptosis in retinoic acid-resistant breast cancer cells . For monitoring resistance development, researchers can use CRABP2 antibodies in longitudinal immunohistochemistry studies of patient samples before treatment and at resistance development to track expression changes correlating with clinical outcomes. In functional studies, combining CRABP2 antibody-based detection with drug sensitivity assays can help identify associations between CRABP2 expression levels and resistance to specific therapies. For mechanistic investigations, use CRABP2 antibodies in ChIP-seq experiments to identify genomic binding sites of CRABP2-associated retinoic acid receptor complexes in sensitive versus resistant cells, potentially revealing altered transcriptional programs. Additionally, immunoprecipitation with CRABP2 antibodies followed by mass spectrometry in sensitive versus resistant cells can identify altered protein interaction networks potentially contributing to resistance. For therapeutic implications, use CRABP2 antibodies to screen for compounds that restore normal CRABP2 function or expression in resistant cells. Finally, combining CRABP2 detection with analysis of ERK/VEGF pathway components may reveal altered signaling dynamics in resistant cells, as these pathways have been linked to CRABP2-mediated cancer progression .

What are the potential applications of CRABP2 antibodies in single-cell analysis of tumor heterogeneity?

CRABP2 antibodies offer significant potential for single-cell analysis of tumor heterogeneity through several cutting-edge methodological approaches. For single-cell protein profiling, researchers can employ mass cytometry (CyTOF) with metal-conjugated CRABP2 antibodies to simultaneously measure CRABP2 expression alongside dozens of other cancer-related proteins at the single-cell level, enabling high-dimensional phenotyping of tumor cells. In spatial context preservation studies, multiplex immunofluorescence or imaging mass cytometry using CRABP2 antibodies alongside other markers can map CRABP2-expressing cells within the tumor microenvironment, providing insights into spatial relationships with stromal components and other cell types. For correlating protein with transcriptomics, CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) can combine CRABP2 antibody detection with single-cell RNA sequencing, revealing relationships between CRABP2 protein levels and transcriptional programs in individual cells. This approach is particularly valuable given findings that CRABP2 expression varies with cancer progression and correlates with prognosis in lung adenocarcinoma and hepatocellular carcinoma . In functional heterogeneity studies, researchers can isolate CRABP2-high versus CRABP2-low cell populations using FACS with CRABP2 antibodies and compare their functional properties including proliferation, migration, invasion, and drug responses. For lineage tracing studies, combining CRABP2 detection with genetic lineage tracing can help determine if CRABP2-expressing cells represent specific tumor subclones with distinct clinical behaviors. These approaches collectively enable researchers to dissect the heterogeneous expression patterns of CRABP2 within tumors and correlate this heterogeneity with functional properties and clinical outcomes.