CSRP2 Antibody Pair

What is CSRP2 and why is it important in research?

CSRP2 is a member of the CSRP family of genes that encode LIM domain proteins involved in regulatory processes critical for development and cellular differentiation. It contains two copies of the cysteine-rich amino acid sequence motif (LIM) with putative zinc-binding activity and plays a role in regulating ordered cell growth . Recent research has revealed CSRP2's significance in cancer biology, particularly its role in suppressing colorectal cancer progression through interaction with p130Cas and inhibition of Rac1 activation . This protein has been studied in several tumor types including breast, gastric, hepatocellular cancers, and lymphoblastic leukemia, though its precise function appears to be context-dependent and requires further investigation . The growing evidence of CSRP2's involvement in crucial cellular processes makes it an important target for research across multiple disciplines including cancer biology, cell differentiation, and development.

How do I select the appropriate anti-CSRP2 antibody for my experiments?

Selecting the appropriate anti-CSRP2 antibody requires careful consideration of several factors including the specific epitope you wish to target, cross-reactivity requirements, and intended applications. First, determine which region of CSRP2 is most relevant to your research question - antibodies targeting different regions (e.g., N-terminal, C-terminal, or specific amino acid sequences like AA 90-139) may yield different results . Second, consider species cross-reactivity - some antibodies show high sequence identity across multiple species (e.g., antibodies with 100% identity to human, gorilla, mouse, rat, and other mammals), which is important for comparative studies . Third, evaluate the validated applications - certain antibodies are specifically validated for Western blotting, while others perform well in IHC, ELISA, or immunofluorescence . For Western blotting, note that the observed molecular weight of CSRP2 is typically 26-30kDa despite a calculated MW of 21kDa, likely due to post-translational modifications . Finally, consider the host species (rabbit, mouse, goat) to ensure compatibility with your experimental design, particularly for co-immunostaining experiments where primary antibody host species must be different.

How should I design co-immunoprecipitation experiments to study CSRP2 protein interactions?

Designing effective co-immunoprecipitation (Co-IP) experiments to study CSRP2 protein interactions requires careful consideration of several critical factors. Based on published methodologies, begin with cell preparation by cultivating cells expressing endogenous CSRP2 or transfected with CSRP2 expression vectors (e.g., HCT116/CSRP2 cells) until approximately 80% confluence . Harvest cells in cold conditions using PBS washes followed by lysis in cold RIPA buffer supplemented with protease and phosphatase inhibitors. After cell lysis, centrifuge at 14,000g at 4°C for 15 minutes to remove cellular debris. To reduce non-specific binding, pre-clear the lysate with appropriate agarose beads (approximately 100 μL protein agarose per 1 mL protein lysate) by incubating with gentle rotation at 4°C for 10 minutes followed by centrifugation . For the immunoprecipitation step, incubate the pre-cleared lysate with anti-CSRP2 antibody overnight at a 4°C with rotation. The next day, add protein A or G agarose beads (depending on the antibody host species) to capture the antigen-antibody complexes and incubate again overnight at 4°C . After thorough washing of the immune complexes with cold lysis buffer, elute the bound proteins by boiling in SDS-PAGE sample buffer. Analyze the immunoprecipitated proteins by Western blotting with antibodies against potential interacting partners. Based on published research, promising interaction candidates include p130Cas, MRTF-A/B, and SRF .

What are the optimal conditions for using anti-CSRP2 antibodies in immunohistochemistry?

Optimizing immunohistochemistry (IHC) conditions for anti-CSRP2 antibody staining requires attention to several critical parameters. Based on established protocols, begin with proper tissue preparation: for paraffin-embedded samples, perform heat-induced antigen retrieval using sodium citrate buffer (pH 6.0) after standard dewaxing and rehydration steps . Block endogenous peroxidase activity with 3% hydrogen peroxide and prevent non-specific binding with 10% FBS in PBS. For primary antibody incubation, use anti-CSRP2 antibody at an optimized dilution (e.g., 1:500 for rabbit anti-CSRP2, HPA045617 from Sigma) and incubate overnight at 4°C in a humidified chamber to ensure consistent staining . Follow with appropriate species-specific secondary antibody (e.g., anti-rabbit IgG at 1:2000 dilution) applied for 40 minutes at 37°C. For visualization, DAB (3,3'-diaminobenzidine) development followed by hematoxylin counterstaining provides excellent contrast for CSRP2 detection. When evaluating staining, be aware that CSRP2 expression varies significantly between tissue types - it shows lower expression in colorectal cancer tissues compared to adjacent normal tissues . Include positive and negative controls in every experiment: normal colon tissue serves as a positive control, while primary antibody omission provides a suitable negative control. For dual immunofluorescence staining to assess co-localization with potential binding partners such as MRTF-A/B or SRF, use fluorophore-conjugated secondary antibodies and confocal microscopy for optimal results .

What protocols should I follow for Western blot detection of CSRP2?

For optimal Western blot detection of CSRP2, follow this comprehensive protocol based on validated research methodologies. Sample preparation is critical: extract total proteins from cells or tissues using RIPA buffer supplemented with protease and phosphatase inhibitors, followed by sonication and centrifugation at 14,000g for 15 minutes at 4°C. Determine protein concentration using a Bradford or BCA assay to ensure equal loading. Load 25μg of protein per lane on a 12% SDS-PAGE gel, as CSRP2 has a molecular weight of 21kDa (calculated) but typically appears at 26-30kDa on Western blots due to post-translational modifications . After separation, transfer proteins to PVDF or nitrocellulose membranes using semi-dry or wet transfer systems (100V for 60-90 minutes). Block membranes with 3-5% nonfat dry milk in TBST for 1 hour at room temperature . Incubate with primary anti-CSRP2 antibody at an optimized dilution (e.g., 1:500 to 1:2000 for the rabbit polyclonal antibody) overnight at 4°C with gentle rocking . After washing with TBST (3 × 10 minutes), incubate with HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG at 1:10,000) for 1 hour at room temperature. Develop using ECL substrate and expose to film or digital imager, with exposure times adjusted based on signal strength (approximately 90 seconds is often sufficient) . For normalization, reprobe with antibodies against housekeeping proteins such as β-actin, GAPDH, or α-tubulin. When troubleshooting, remember that CSRP2 expression varies across cell lines and tissues, with notably lower expression in colorectal cancer tissues compared to normal adjacent tissues .

How can I use CSRP2 antibodies to investigate its role in cancer progression?

Investigating CSRP2's role in cancer progression requires a multifaceted approach using various antibody-dependent techniques. Begin with expression profiling across different cancer types and stages using immunohistochemistry on tissue microarrays with validated anti-CSRP2 antibodies (e.g., rabbit anti-CSRP2, HPA045617) . Compare CSRP2 expression levels between tumor and adjacent normal tissues, noting that CSRP2 shows decreased expression in colorectal cancer tissues compared to normal tissues, indicating a potential tumor suppressor role in this context . For mechanistic studies, employ co-immunoprecipitation to identify CSRP2 interaction partners in cancer cell lines, focusing on the CSRP2/p130Cas/Rac1 axis previously implicated in colorectal cancer inhibition . Combine this with Western blotting to monitor changes in EMT markers and activation status of Hippo, ERK, and PAK signaling pathways following CSRP2 knockdown or overexpression. For functional studies, establish stable cell lines with modulated CSRP2 expression using lentiviral constructs and validate expression changes by Western blotting with anti-CSRP2 antibodies. Use these cell lines in proliferation, migration, and invasion assays to determine how CSRP2 influences cancer cell behavior . Additionally, employ immunofluorescence with anti-CSRP2 antibodies to examine subcellular localization changes during cancer progression, particularly focusing on potential co-localization with actin cytoskeleton components, as CSRP2 contributes to actin backbone assembly and maintenance . Finally, use chromatin immunoprecipitation (ChIP) assays to investigate how CSRP2 influences gene expression through its interaction with transcription factors like SRF and MRTF-A/B .

What is the significance of CSRP2-MRTF interaction in myofibroblast function?

The CSRP2-MRTF interaction represents a critical regulatory mechanism in myofibroblast function with significant implications for fibrotic disorders and tissue remodeling. CSRP2 (Cysteine and Glycine-Rich Protein 2) directly binds to myocardin-related transcription factors (MRTF-A/B) and serum response factor (SRF), stabilizing the MRTF/SRF/CArG-box complex . This interaction is functionally significant as it influences the transcriptional program controlling myofibroblast differentiation and activity. Myofibroblasts, characterized by expression of α-smooth muscle actin (α-SMA), play crucial roles in wound healing, fibrosis, and cancer progression through their contractile properties and extracellular matrix production. To investigate this interaction experimentally, researchers should employ co-immunoprecipitation using anti-CSRP2 and anti-MRTF-A/B antibodies to confirm the physical interaction between these proteins in relevant cell types . Chromatin immunoprecipitation (ChIP) assays with anti-MRTF-A antibodies can further elucidate how this interaction affects binding to CArG-box-containing promoters of myofibroblast marker genes. Functional studies should include assessment of myofibroblast differentiation markers (α-SMA, collagen type I) by Western blotting and immunofluorescence following modulation of CSRP2 expression. Actin cytoskeleton dynamics, which are central to MRTF regulation, can be evaluated using phalloidin staining and analyzing cofilin phosphorylation status in the presence of actin-modulating compounds like Latrunculin B or Rho-associated protein kinase inhibitor Y27632 . Understanding this interaction pathway provides insights into potential therapeutic targets for conditions characterized by myofibroblast dysregulation.

How can I differentiate between CSRP2 and other CSRP family members in my experiments?

Differentiating between CSRP2 and other CSRP family members (CSRP1 and CSRP3) requires careful antibody selection and experimental design to ensure specificity. First, select antibodies that target non-conserved regions of the protein. While all CSRP family members contain LIM domains with high sequence similarity, they differ significantly in certain regions, particularly between the LIM domains and at the C-terminus . When selecting commercial antibodies, examine the immunogen information carefully - antibodies raised against recombinant full-length CSRP2 or peptides corresponding to unique regions (e.g., amino acids 1-193 of human CSRP2) offer better specificity than those targeting conserved LIM domains . Perform validation experiments using positive and negative controls: cell lines or tissues with documented differential expression of CSRP family members can serve as controls. For instance, vascular smooth muscle cells express CSRP2 at detectable levels . Always include specificity controls in Western blotting by running samples from cells overexpressing each CSRP family member side-by-side to confirm band size differences and antibody specificity. For immunohistochemistry or immunofluorescence, perform peptide competition assays where the antibody is pre-incubated with excess target peptide to confirm staining specificity. Additionally, use RNA interference targeting each family member individually to verify antibody specificity - the signal should decrease only when the targeted family member is knocked down. At the RNA level, design PCR primers that amplify unique regions, such as the forward primer 5'-TCACGATGAAGAGATCTACTGC-3' and reverse primer 5'-AGTGTTTGGATTTGTTGTAGGC-3' used for CSRP2 amplification in published studies .

Why might I observe different molecular weights for CSRP2 in Western blot analysis?

Observing different molecular weights for CSRP2 in Western blot analysis is a common challenge that can be attributed to several biological and technical factors. The calculated molecular weight of CSRP2 is 21kDa, but it typically appears at 26-30kDa on Western blots . This discrepancy primarily stems from post-translational modifications, particularly phosphorylation events that affect protein mobility during electrophoresis. CSRP2 contains multiple potential phosphorylation sites that can be differentially modified depending on cellular context or signaling status. Alternative splicing also contributes to size variations - CSRP2 gene can undergo alternative splicing resulting in multiple transcript variants with different protein products . When troubleshooting variable molecular weights, first verify antibody specificity using positive controls (recombinant CSRP2 protein) and negative controls (CSRP2 knockout or knockdown samples). Technical variables can also contribute to apparent molecular weight differences: different percentage gels significantly affect protein migration patterns (use 12-15% gels for optimal resolution of smaller proteins like CSRP2), and variations in sample preparation methods, particularly buffer composition and reducing agent concentration, can affect protein denaturation and apparent size. To distinguish between true isoforms and technical artifacts, treat samples with phosphatase before electrophoresis - if higher molecular weight bands collapse to the predicted 21kDa size, this confirms phosphorylation as the cause. Additionally, different antibodies targeting different epitopes (N-terminal vs. C-terminal) may preferentially recognize certain modified forms or splice variants of CSRP2, resulting in apparently different banding patterns.

What controls should I include when validating a new anti-CSRP2 antibody?

Validating a new anti-CSRP2 antibody requires a comprehensive set of controls to ensure specificity, sensitivity, and reproducibility across experimental applications. First, include positive controls: cell lines or tissues with confirmed high CSRP2 expression (based on published literature) or recombinant CSRP2 protein at known concentrations. For negative controls, use CSRP2 knockout models generated through CRISPR-Cas9, cells treated with validated CSRP2-specific siRNA/shRNA, or tissues known to have minimal CSRP2 expression. To confirm specificity, perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide or recombinant CSRP2 protein before application - this should abolish specific signal while leaving non-specific binding unaffected . Cross-reactivity assessment is essential since CSRP2 belongs to a family of similar proteins (CSRP1, CSRP3) - test the antibody against recombinant proteins of all family members and in cells overexpressing each family member individually. For application-specific validation, include appropriate technical controls: for Western blotting, verify that the observed molecular weight matches the expected size range (26-30kDa) and include loading controls (β-actin, GAPDH) ; for IHC, include isotype controls using non-specific IgG from the same host species at the same concentration as the CSRP2 antibody; for immunofluorescence, include secondary-only controls to assess background fluorescence. To validate across species, test the antibody on samples from different species if cross-reactivity is claimed, particularly important since CSRP2 shows high sequence conservation across many species (human, mouse, rat, bovine, etc.) . Document all validation results, including images of Western blots showing full membranes, representative IHC/IF images with controls, and quantitative assessments of specificity and sensitivity for reference in future experiments.

How can CSRP2 antibodies be used to study cytoskeletal dynamics?

CSRP2 antibodies offer powerful tools for investigating cytoskeletal dynamics due to CSRP2's established role in actin cytoskeleton organization. Previous studies have demonstrated that CSRP2 contributes to the assembly and maintenance of the actin backbone and is involved in actin cytoskeleton rearrangement . To study these dynamics, researchers can employ several antibody-dependent approaches. Immunofluorescence co-staining with anti-CSRP2 antibodies and fluorescently labeled phalloidin (which binds F-actin) enables visualization of CSRP2's spatial relationship with the actin cytoskeleton under various conditions . Live-cell imaging using fluorescently tagged CSRP2 constructs (validated by immunoblotting with anti-CSRP2 antibodies) allows real-time observation of CSRP2's dynamic localization during cell migration, division, or in response to cytoskeletal-disrupting agents like Latrunculin B . For biochemical analyses, co-immunoprecipitation with anti-CSRP2 antibodies followed by mass spectrometry can identify novel CSRP2-interacting cytoskeletal proteins. Additionally, proximity ligation assays using anti-CSRP2 antibodies paired with antibodies against known cytoskeletal regulators (e.g., cofilin, cortactin) can reveal direct interactions within intact cells . In functional studies, researchers can manipulate CSRP2 expression (verified by Western blotting) and examine effects on cytoskeletal parameters including F/G-actin ratio, focal adhesion dynamics, and cell contractility. Scanning electron microscopy combined with immunogold labeling using anti-CSRP2 antibodies provides ultra-high-resolution visualization of CSRP2's association with specific cytoskeletal structures . These approaches can be particularly valuable in contexts where cytoskeletal dynamics are critical, such as cancer cell invasion, wound healing, and developmental processes.

What is the potential of CSRP2 as a biomarker in cancer prognosis?

CSRP2's potential as a biomarker in cancer prognosis represents an emerging area of research with significant clinical implications. Evidence supporting CSRP2's biomarker potential comes from studies demonstrating that CSRP2 expression levels in colorectal cancer (CRC) tissues are significantly lower than in adjacent normal tissues and correlate with poor prognosis in CRC patients . This prognostic relationship appears to be mechanistically grounded in CSRP2's function as a suppressor of epithelial-mesenchymal transition (EMT) and metastasis through inhibition of the p130Cas/Rac1 axis and regulation of Hippo, ERK, and PAK signaling pathways . To investigate CSRP2's biomarker potential, researchers should conduct large-scale immunohistochemical analyses of tissue microarrays using validated anti-CSRP2 antibodies, correlating expression levels with clinicopathological parameters and patient outcomes through Kaplan-Meier survival analysis . Standardization of CSRP2 detection is critical - researchers should establish scoring systems based on staining intensity and percentage of positive cells, validated across multiple cohorts. Multivariate analyses incorporating CSRP2 expression alongside established prognostic factors can determine its independent prognostic value. Beyond CRC, researchers should evaluate CSRP2's prognostic significance in other cancer types where its role remains controversial, including breast, gastric, and hepatocellular cancers . For translational relevance, development of CSRP2-based liquid biopsy approaches could enable non-invasive monitoring of disease progression. Additionally, investigating the relationship between CSRP2 expression and treatment response could reveal its potential as a predictive biomarker for specific therapeutic strategies, particularly those targeting metastasis or EMT.

How do post-translational modifications affect CSRP2 function and antibody detection?

Post-translational modifications (PTMs) significantly impact both CSRP2 function and antibody-based detection methods, creating important considerations for research design and data interpretation. While specific PTMs of CSRP2 are not extensively characterized in the provided search results, the discrepancy between its calculated molecular weight (21kDa) and observed Western blot size (26-30kDa) strongly suggests the presence of substantial modifications . Phosphorylation likely plays a key role in CSRP2 regulation, particularly given its involvement in signaling pathways like Hippo, ERK, and PAK/LIMK/cortactin . These phosphorylation events may modulate CSRP2's interaction with binding partners such as p130Cas, MRTF-A/B, and SRF, thereby affecting its function in transcriptional regulation and cytoskeletal organization . For investigating PTMs, researchers should employ phospho-specific antibodies when available or use general phospho-protein detection methods (e.g., Pro-Q Diamond staining) followed by identification with standard anti-CSRP2 antibodies. Mass spectrometry analysis of immunoprecipitated CSRP2 can map specific modification sites. The choice of antibody is critical when studying CSRP2 PTMs - epitope-specific antibodies may have differential reactivity to modified forms of CSRP2. For instance, antibodies targeting regions containing potential phosphorylation sites may show reduced binding when those sites are phosphorylated. When conflicting results arise from different anti-CSRP2 antibodies, researchers should consider whether the disparities reflect detection of differently modified CSRP2 populations rather than experimental inconsistencies. Treatment with phosphatases, deubiquitinases, or other PTM-removing enzymes before analysis can help determine which modifications affect antibody recognition. Additionally, researchers should investigate how PTMs affect CSRP2 subcellular localization, stability, and function under different cellular conditions (e.g., growth factor stimulation, mechanical stress) to better understand their physiological relevance.

How can I design multiplexed immunoassays to study CSRP2 in complex signaling networks?

Designing multiplexed immunoassays to study CSRP2 within complex signaling networks requires careful consideration of antibody compatibility, detection methods, and experimental controls. Begin by mapping the relevant signaling networks based on current knowledge - CSRP2 interacts with p130Cas and influences the Hippo, ERK, and PAK/LIMK/cortactin signaling pathways , while also binding to MRTF-A/B and SRF to affect transcriptional regulation . For multiplex immunofluorescence, select primary antibodies raised in different host species (e.g., rabbit anti-CSRP2 and mouse anti-p130Cas) to enable simultaneous detection with species-specific secondary antibodies. Tyramide signal amplification can overcome sensitivity limitations when detecting low-abundance proteins within the network. For multiparameter flow cytometry, conjugate anti-CSRP2 antibodies directly to fluorophores with non-overlapping emission spectra from those used for other pathway components. In both approaches, include appropriate controls: single-stained samples to establish compensation parameters, isotype controls for each antibody, and biological controls (e.g., cells with CSRP2 knockdown or overexpression). Proximity ligation assays offer another powerful approach for detecting protein-protein interactions involving CSRP2 within intact cells - pairs of antibodies against CSRP2 and potential interaction partners will generate fluorescent signals only if the proteins are within 40nm of each other . For high-throughput analysis, reverse phase protein arrays or multiplex bead-based assays can simultaneously measure CSRP2 and multiple signaling proteins across many samples. To study dynamic changes in signaling networks, design time-course experiments with stimulation by relevant factors like TGF-β2, which affects myofibroblast differentiation involving CSRP2-MRTF interactions . Finally, apply computational methods like principal component analysis or partial least squares regression to interpret the complex multidimensional data generated by these approaches, revealing how CSRP2 integrates into broader signaling networks.

What is the relationship between CSRP2 gene variants and protein function across different cell types?

The relationship between CSRP2 gene variants and protein function across different cell types represents a complex but important area for investigation, though detailed information on this topic is limited in the provided search results. CSRP2 undergoes alternative splicing resulting in multiple transcript variants , suggesting potential functional diversity across different cell types and physiological contexts. To systematically investigate this relationship, researchers should begin with comprehensive expression profiling of CSRP2 splice variants across diverse cell types using RNA sequencing and isoform-specific PCR techniques, designed with primers like those previously validated for CSRP2 amplification (5'-TCACGATGAAGAGATCTACTGC-3' forward and 5'-AGTGTTTGGATTTGTTGTAGGC-3' reverse) . For protein-level characterization, use antibodies targeting conserved regions to detect all variants, complemented by variant-specific antibodies when available. Functional studies should include overexpression of specific CSRP2 variants in relevant cell types, followed by phenotypic assays examining cytoskeletal organization, cell migration, and transcriptional activity. Cell type-specific functions may be particularly evident when comparing mesenchymal cells (where CSRP2's role in cytoskeletal organization may predominate) versus epithelial cells (where its tumor suppressor functions may be more relevant) . The contradictory roles of CSRP2 observed across different cancer types suggest cell-specific binding partners or regulatory mechanisms . Co-immunoprecipitation studies using anti-CSRP2 antibodies followed by mass spectrometry can identify cell type-specific interaction partners that may explain functional differences. Additionally, researchers should investigate epigenetic regulation of CSRP2 variants across cell types using chromatin immunoprecipitation and DNA methylation analysis. For translational relevance, correlate specific CSRP2 variants with clinical outcomes in diseases where CSRP2 has been implicated, such as colorectal cancer, potentially revealing variant-specific prognostic value .