CSRP1 Antibody

What is CSRP1 and what are its primary functions in cellular biology?

CSRP1 (also known as CRP, CRP1, CSRP, or CYRP) is a member of the cysteine and glycine-rich protein family that plays crucial roles in cell adhesion and cytoskeletal organization . Research suggests it significantly contributes to cell morphology and migration processes, making it essential for tissue development and wound healing . Additionally, CSRP1 may have important functions in neuronal development . The protein contains specific structural domains that facilitate its interaction with other cellular components, particularly those involved in cytoskeletal dynamics. Its regulatory functions appear to be context-dependent, varying across different tissue types and developmental stages.

How are CSRP1 antibodies produced, and what types are available for research?

CSRP1 antibodies are typically generated by immunizing host animals (commonly rabbits) with either recombinant fusion proteins containing specific amino acid sequences of human CSRP1 or with synthesized peptides derived from CSRP1 protein . The most common types available include:

• Polyclonal antibodies: Generated in rabbits against various epitopes of CSRP1, these antibodies recognize multiple regions of the target protein. For example, the CAB1071 antibody is produced using a recombinant fusion protein corresponding to amino acids 1-193 of human CSRP1 (NP_004069.1) .

• Monoclonal antibodies: Produced from single B-cell clones, providing higher specificity for particular epitopes. Some monoclonal antibodies like clone 2A11 are available for CSRP1 detection .

Antibodies targeting different regions of the CSRP1 protein are available, including those recognizing the internal region (AA 51-100, AA 71-120) and broader regions (AA 1-193, AA 94-192) . These variations allow researchers to select antibodies appropriate for specific experimental requirements and applications.

What validation methods should be employed to confirm CSRP1 antibody specificity?

To ensure experimental rigor, CSRP1 antibody specificity should be validated through multiple complementary approaches:

• Western blotting with positive control samples (such as A-549, BT-474, HeLa cells for human samples, or appropriate mouse tissues) . Compare the observed molecular weight with the expected size of CSRP1 (~21 kDa).

• Immunoprecipitation followed by mass spectrometry to confirm pull-down of authentic CSRP1 protein.

• RNA interference or CRISPR knockout validation, where CSRP1 expression is reduced/eliminated and corresponding reduction in antibody signal is observed.

• Cross-reactivity testing against related proteins, particularly other CSRP family members like CSRP2 and CSRP3, to ensure specificity.

• Immunohistochemistry with appropriate positive and negative control tissues, comparing staining patterns with known CSRP1 expression profiles.

• Peptide competition assays, where pre-incubation with the immunizing peptide should abolish specific antibody binding.

These validation steps are critical for preventing experimental artifacts and ensuring reliable research outcomes.

What are the optimal conditions for using CSRP1 antibodies in Western blotting?

For optimal Western blotting results with CSRP1 antibodies, researchers should consider the following protocol:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Load 20-30 μg of total protein per lane

  • Include positive control samples (e.g., A-549, BT-474, HeLa cells)

• Protein separation:

  • Use 12-15% SDS-PAGE gels (optimal for visualizing CSRP1's ~21 kDa band)

  • Run at 100-120V for adequate separation

• Transfer conditions:

  • Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

  • Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute primary CSRP1 antibody at 1:500 to 1:2000 in blocking solution

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-5 times with TBST (5 minutes each)

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Wash 3-5 times with TBST

• Detection:

  • Use enhanced chemiluminescence detection

  • Typical exposure times range from 30 seconds to 5 minutes

The antibody should detect a single band at approximately 21 kDa, corresponding to CSRP1 protein.

How can CSRP1 antibodies be optimized for immunohistochemistry (IHC) applications?

Optimization of CSRP1 antibodies for IHC requires careful consideration of several parameters:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) sections (4-6 μm thickness)

  • Freshly cut sections yield better results than stored slides

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow cooling to room temperature (approximately 20 minutes)

• Blocking steps:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Block non-specific binding with 5-10% normal serum from secondary antibody host species

  • Consider additional blocking if background is observed (e.g., avidin/biotin blocking)

• Antibody dilution and incubation:

  • Test a range of antibody dilutions (start with manufacturer recommendations)

  • Incubate primary antibody overnight at 4°C in a humidified chamber

  • Use appropriate detection systems (e.g., polymer-based or ABC method)

• Controls:

  • Always include positive control tissues with known CSRP1 expression

  • Include negative controls (omitting primary antibody)

  • Consider using tissues from CSRP1 knockout models as specificity controls

• Counterstaining and mounting:

  • Light hematoxylin counterstaining provides optimal nuclear detail without obscuring CSRP1 staining

  • Use permanent mounting media for long-term storage

With proper optimization, CSRP1 antibodies can provide specific staining in various tissue types, enabling both research and potential diagnostic applications.

What approaches can be used to study CSRP1 protein-protein interactions?

Several complementary approaches can be employed to investigate CSRP1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4 μg of CSRP1 antibody per 200-400 μg of cell/tissue extract

  • Pre-clear lysates with Protein A/G beads before antibody addition

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

  • Analyze interacting partners by Western blotting or mass spectrometry

• RNA immunoprecipitation (RIP):

  • For studying RNA-protein interactions with CSRP1

  • Use dilutions of 1:20 to 1:50 of CSRP1 antibody

  • Follow with RT-qPCR or RNA-seq to identify bound transcripts

• Proximity ligation assay (PLA):

  • Visualize protein-protein interactions in situ

  • Requires CSRP1 antibody raised in a different host species than antibodies against potential interacting partners

  • Provides spatial information about interaction sites within cells

• Protein-protein interaction network analysis:

  • Build on experimental data to construct interaction networks

  • Similar to the approach used in the CSRP1 study in AML where 19 genes were identified in the most significant module of the protein-protein interaction network

• Yeast two-hybrid or mammalian two-hybrid screening:

  • For discovery of novel CSRP1 interacting partners

  • Requires construction of CSRP1 fusion proteins as "bait"

These methods, used individually or in combination, can provide comprehensive insights into the interactome of CSRP1 and its functional relevance in different cellular contexts.

How does CSRP1 expression correlate with clinical outcomes in acute myeloid leukemia?

Research has established significant correlations between CSRP1 expression and clinical outcomes in AML patients:

• Survival impact: High CSRP1 expression is associated with significantly worse prognosis in AML patients, with a hazard ratio of 2.36 (95% CI: 1.53-3.64; P < 0.001) in the TCGA-LAML dataset. This finding has been validated across multiple independent cohorts, including the Beat-AML dataset, ZZU cohort, and GSE12417 dataset .

• Multivariate analysis: CSRP1 remains an independent prognostic factor after adjusting for established risk factors, with an adjusted HR of 2.048 (95% CI: 1.313-3.193; P = 0.002) .

• Clinical characteristics: The table below summarizes key clinical differences between high and low CSRP1 expression groups in AML:

• Molecular associations: High CSRP1 expression correlates with increased frequency of DNMT3A mutations .

• Predictive value: Time-dependent ROC curve analysis from the TCGA-LAML dataset demonstrates excellent predictive performance of CSRP1 for AML patient survival .

These findings suggest that CSRP1 expression analysis, facilitated by antibody-based detection methods, may have utility in AML risk stratification and treatment decision-making.

What molecular mechanisms might explain CSRP1's role in cancer progression?

Several potential molecular mechanisms may explain CSRP1's role in cancer progression:

• Cytoskeletal regulation and cell migration:

  • CSRP1's established role in cytoskeletal organization and cell morphology suggests it may influence cancer cell migration and invasion

  • Changes in cell adhesion mediated by CSRP1 could affect metastatic potential

• Signaling pathway modulation:

  • Functional enrichment analysis of CSRP1-related differentially expressed genes in AML revealed associations with neutrophil activation and apoptosis pathways

  • This suggests CSRP1 may influence cancer cell survival and immune evasion mechanisms

• Drug sensitivity correlations:

  • High CSRP1 expression in AML correlates with higher sensitivity to certain chemotherapeutic agents (5-fluorouracil, gemcitabine, rapamycin, cisplatin) but lower sensitivity to fludarabine

  • This implies CSRP1 may modulate drug response pathways

• Epigenetic regulation:

  • In hepatocellular carcinoma, abnormal methylation inactivates CSRP1, suggesting epigenetic control of its expression is important in some cancer contexts

  • CSRP1's association with DNMT3A mutations in AML further suggests connections to epigenetic mechanisms

• Tissue-specific effects:

  • CSRP1 shows differential expression across cancer types (overexpressed in 7 cancers, underexpressed in 18)

  • This suggests context-dependent functions potentially related to tissue-specific interacting partners

Understanding these mechanisms requires sophisticated experimental approaches using CSRP1 antibodies for protein detection, localization, and interaction studies. Further research may reveal novel therapeutic opportunities targeting CSRP1 or its associated pathways.

How can CSRP1 antibodies be utilized to investigate differential expression across cancer types?

CSRP1 antibodies can be strategically employed to investigate differential expression across cancer types through several methodological approaches:

• Tissue microarray (TMA) analysis:

  • Utilize CSRP1 antibodies for immunohistochemical staining of TMAs containing multiple cancer types

  • Quantify staining intensity and proportion of positive cells using digital pathology platforms

  • Correlate expression patterns with clinicopathological features and survival data

  • This approach could validate the RNA-seq findings showing CSRP1 overexpression in 7 cancers and underexpression in 18 others

• Western blot comparative analysis:

  • Analyze CSRP1 protein levels across cancer cell line panels representing diverse tumor types

  • Standardize loading with appropriate housekeeping proteins (β-actin, GAPDH)

  • Quantify relative expression levels through densitometry

  • Use dilutions of 1:500 to 1:2000 for optimal detection

• Flow cytometry for cancer cell population analysis:

  • Develop protocols for intracellular staining with CSRP1 antibodies

  • Analyze heterogeneity within tumor samples at the single-cell level

  • Combine with other markers to identify specific cellular subsets with altered CSRP1 expression

• Subcellular localization studies:

  • Use immunofluorescence with CSRP1 antibodies to determine whether subcellular localization differs across cancer types

  • Co-stain with markers for specific cellular compartments to assess potential functional differences

• Comparative phosphoproteomic analysis:

  • Develop or obtain phospho-specific CSRP1 antibodies

  • Investigate whether post-translational modifications of CSRP1 differ between cancer types

  • Correlate modification patterns with activation of specific signaling pathways

These approaches allow researchers to move beyond transcriptomic data to validate protein-level differences, potentially revealing new diagnostic markers or therapeutic targets across cancer types.

What are common challenges when using CSRP1 antibodies, and how can they be addressed?

Researchers frequently encounter several challenges when working with CSRP1 antibodies that can be systematically addressed:

• Non-specific binding and background:

  • Problem: High background or multiple bands in Western blots

  • Solutions:

    • Increase blocking time/concentration (try 5% BSA instead of milk for phospho-epitopes)

    • Optimize antibody dilution (test range from 1:500 to 1:2000)

    • Increase washing duration and frequency

    • Pre-adsorb antibody with non-specific proteins

    • Use more stringent washing buffers (increase Tween-20 concentration slightly)

• Inconsistent immunoprecipitation results:

  • Problem: Poor or variable pull-down efficiency

  • Solutions:

    • Adjust antibody amount (0.5-4μg per 200-400μg extract)

    • Pre-clear lysates thoroughly

    • Extend incubation time

    • Optimize lysis buffer composition to preserve protein-protein interactions

    • Consider crosslinking antibody to beads to prevent co-elution

• Poor signal in immunohistochemistry:

  • Problem: Weak or absent staining

  • Solutions:

    • Test multiple antigen retrieval methods (citrate vs. EDTA buffers)

    • Extend primary antibody incubation time

    • Use amplification systems (e.g., tyramide signal amplification)

    • Ensure tissue fixation is optimal (overfixation can mask epitopes)

    • Try different CSRP1 antibodies targeting different epitopes

• Cross-reactivity with CSRP family members:

  • Problem: Antibody may detect related proteins (CSRP2, CSRP3)

  • Solutions:

    • Verify antibody specificity using recombinant CSRP proteins

    • Use knockout/knockdown controls

    • Select antibodies targeting unique regions of CSRP1

    • Confirm results with orthogonal methods

• Batch-to-batch variability:

  • Problem: Inconsistent results between antibody lots

  • Solutions:

    • Purchase larger quantities of single lots for long-term projects

    • Validate each new lot against previous lots

    • Maintain reference standards and positive controls

    • Consider monoclonal antibodies for greater consistency

Applying these systematic troubleshooting approaches can significantly improve experimental outcomes with CSRP1 antibodies.

How should researchers optimize CSRP1 antibody-based protocols for different sample types?

Optimization strategies for CSRP1 antibody protocols should be tailored to specific sample types:

• Cell lines:

  • Lysis buffer: Standard RIPA buffer works well for most applications

  • Protein amount: 20-30μg for Western blot

  • Fixation for ICC: 4% paraformaldehyde (10 minutes) preserves cytoskeletal elements

  • Antibody dilution: Start with 1:1000 for Western blots and 1:200 for ICC

  • Positive controls: A-549, BT-474, and HeLa cells show reliable CSRP1 expression

• Primary tissue samples:

  • Lysis buffer: Consider gentler options like NP-40 buffer to preserve protein complexes

  • Homogenization: Optimize mechanical disruption to maintain protein integrity

  • Protein amount: Increase to 40-50μg for Western blots due to cellular heterogeneity

  • Fixation for IHC: 10% neutral buffered formalin (24-48 hours) followed by careful paraffin embedding

  • Antibody dilution: May require higher concentrations (1:100-1:500)

  • Controls: Include adjacent normal tissue when available

• Bone marrow samples (relevant for AML research):

  • Sample preparation: Red blood cell lysis before protein extraction

  • Protein isolation: Consider nuclear/cytoplasmic fractionation

  • Antibody concentration: May need higher concentrations (1:200-1:500)

  • Background reduction: Extended blocking (2 hours) to minimize non-specific binding

  • Controls: Compare with normal bone marrow samples

  • Applications: Flow cytometry protocols can be developed for analyzing CSRP1 in specific cell populations

• Liquid biopsies (serum/plasma):

  • Pre-clearing: Remove abundant proteins (albumin, immunoglobulins) before analysis

  • Concentration: May require sample concentration techniques

  • Detection methods: Consider using more sensitive detection methods (e.g., chemiluminescent substrates with extended exposure)

  • Validation: Always confirm with cellular/tissue samples when possible

• Mouse models:

  • Cross-reactivity: Confirm antibody reactivity with mouse CSRP1

  • Tissue processing: Optimize fixation time based on specific tissues

  • Background reduction: Use mouse-on-mouse blocking kits if using mouse monoclonal antibodies

  • Controls: Include tissues from CSRP1 knockout mice when available

Each sample type presents unique challenges requiring systematic optimization to achieve reliable and reproducible results with CSRP1 antibodies.

What novel applications of CSRP1 antibodies might advance our understanding of disease mechanisms?

Several innovative applications of CSRP1 antibodies could significantly advance our understanding of disease mechanisms:

• Single-cell protein analysis:

  • Apply CSRP1 antibodies in mass cytometry (CyTOF) or imaging mass cytometry

  • Investigate heterogeneity of CSRP1 expression at single-cell resolution within tumors

  • Correlate with other markers to identify specific cellular subpopulations with unique CSRP1 expression patterns

  • This could reveal previously unrecognized cellular states in diseases like AML

• Proximity-dependent labeling:

  • Use CSRP1 antibodies conjugated to enzymes like APEX2 or BioID

  • Map the proximal proteome around CSRP1 in different cellular contexts

  • Identify context-specific interaction networks that could explain differential functions across tissue types

  • This approach could elucidate why CSRP1 shows opposing expression patterns across cancer types

• Spatial transcriptomics combined with protein detection:

  • Integrate in situ hybridization with immunofluorescence using CSRP1 antibodies

  • Correlate protein expression with transcriptional profiles in the spatial context of tissues

  • Map CSRP1 expression to specific tissue microenvironments

  • This could reveal niche-dependent regulation of CSRP1 in development and disease

• Antibody-based therapeutic targeting:

  • Develop conjugated antibodies targeting CSRP1 for potential therapeutic applications

  • Explore selective delivery of cytotoxic agents to CSRP1-overexpressing AML cells

  • Investigate whether CSRP1-targeting can sensitize cells to conventional therapies

  • This translational approach could leverage the finding that high CSRP1 expression predicts poor outcomes in AML

• Mechanisms of drug sensitivity modulation:

  • Use CSRP1 antibodies to track changes in expression, localization, and interactions following drug treatment

  • Investigate the molecular basis for differential drug sensitivity (e.g., higher sensitivity to 5-fluorouracil but lower sensitivity to fludarabine)

  • Develop predictive models for personalized therapeutic approaches based on CSRP1 status

These novel applications could transform our understanding of CSRP1's role in disease and potentially lead to new diagnostic and therapeutic strategies.

How might CSRP1 antibodies be integrated into clinical diagnostics for cancer patients?

The integration of CSRP1 antibodies into clinical diagnostics represents a promising frontier for improving cancer patient care:

• Prognostic and predictive biomarker development:

  • Standardize immunohistochemical protocols for CSRP1 detection in diagnostic pathology

  • Develop scoring systems correlating CSRP1 expression levels with clinical outcomes

  • Create predictive algorithms incorporating CSRP1 status alongside established risk factors

  • This could enhance current risk stratification systems for AML, which research shows "remain insufficient to select the best schedules"

• Companion diagnostics for targeted therapies:

  • Develop CSRP1 antibody-based assays to identify patients likely to respond to specific treatments

  • Leverage findings that high CSRP1 expression correlates with differential drug sensitivities

  • Create standardized testing platforms suitable for clinical laboratory implementation

  • Validate cutoff values for treatment decisions in prospective clinical trials

• Minimal residual disease (MRD) monitoring:

  • Develop highly sensitive flow cytometry protocols using CSRP1 antibodies

  • Create multiplexed panels incorporating CSRP1 alongside established MRD markers

  • Evaluate whether CSRP1 detection can improve MRD sensitivity in AML

  • This application could be particularly valuable given CSRP1's association with poor prognosis

• Liquid biopsy applications:

  • Develop techniques to detect CSRP1 protein in circulating tumor cells or extracellular vesicles

  • Create sensitive immunoassays for CSRP1 detection in blood samples

  • Correlate circulating CSRP1 levels with disease status and treatment response

  • This non-invasive approach could facilitate longitudinal monitoring

• Multiparameter diagnostic platforms:

  • Integrate CSRP1 antibodies into multiplexed immunofluorescence panels

  • Combine with other prognostic markers for comprehensive tumor profiling

  • Develop image analysis algorithms to quantify CSRP1 expression patterns

  • This approach could capture the complex biological context in which CSRP1 functions

Implementation of these approaches would require rigorous clinical validation, standardization across laboratories, and demonstration of clinical utility beyond existing diagnostic methods.

How can computational approaches enhance CSRP1 antibody-based research in precision medicine?

Computational approaches can significantly enhance CSRP1 antibody-based research, particularly in advancing precision medicine:

• Automated image analysis for immunohistochemistry:

  • Develop deep learning algorithms for quantitative assessment of CSRP1 staining

  • Enable standardized scoring across laboratories

  • Identify subtle staining patterns not apparent to human observers

  • Integrate with digital pathology platforms for clinical implementation

  • This approach could standardize the prognostic value of CSRP1 in AML and other cancers

• Multi-omics data integration:

  • Correlate CSRP1 protein levels (detected by antibodies) with transcriptomic, genomic, and epigenomic data

  • Build predictive models incorporating CSRP1 status with other molecular features

  • Identify patient subgroups with distinct biological and clinical characteristics

  • Similar to methods used in the study that identified correlations between CSRP1 and DNMT3A mutations

• Network-based analyses:

  • Expand protein-protein interaction networks identified through CSRP1 antibody-based methods

  • Apply graph theory algorithms to identify critical nodes and potential therapeutic targets

  • Integrate with knowledge bases of biological pathways

  • This could build upon the PPI network analysis that identified 19 genes in the most significant module associated with CSRP1 in AML

• Drug sensitivity prediction:

  • Develop machine learning models using CSRP1 antibody-based assay results to predict therapeutic responses

  • Implement algorithms similar to the OncoPredict algorithm used to explore correlations between CSRP1 and drug resistance

  • Create decision support tools for clinicians selecting treatment regimens

  • This approach could leverage findings that high CSRP1 expression correlates with differential drug sensitivities

• Virtual screening for targeted therapeutics:

  • Use structural data and protein interaction networks identified through antibody-based methods

  • Apply molecular docking simulations to identify compounds targeting CSRP1 or its interaction partners

  • Prioritize candidate compounds for experimental validation

  • This approach could advance the suggestion that "CSRP1 may serve as a potential therapeutic target for AML in the future"