SRGAP1 Antibody
Description
Introduction to SRGAP1 Antibodies
SRGAP1 antibodies are immunological reagents specifically designed to detect and bind to SRGAP1 protein, also known as ARHGAP13, which functions as a GTPase-activating protein (GAP) for Rho-family GTPases. These antibodies have become indispensable tools for researchers investigating cell migration, neuronal development, and cancer progression . The development of various SRGAP1 antibody formats has enabled multiple analytical approaches, from protein quantification to localization studies within complex cellular environments.
SRGAP1 antibodies are commercially available in multiple formats, including monoclonal and polyclonal variants, with different species origins and conjugation options to suit diverse experimental needs. Their high specificity and versatility make them valuable research tools across multiple disciplines, including oncology, neurobiology, and cell biology .
Monoclonal SRGAP1 Antibodies
Monoclonal SRGAP1 antibodies, such as the D-11 variant, are produced from single B-cell clones, resulting in antibodies with identical specificity. The D-11 antibody is a mouse monoclonal IgG2a with kappa light chain that specifically recognizes SRGAP1 protein from multiple species, including human, mouse, and rat . This antibody has been validated for multiple applications, ensuring reliable detection of SRGAP1 across different experimental platforms.
Polyclonal SRGAP1 Antibodies
Polyclonal SRGAP1 antibodies, like those produced by Novus Biologicals, are typically developed in rabbits immunized with recombinant SRGAP1 protein fragments. These antibodies recognize multiple epitopes on the SRGAP1 protein, potentially offering enhanced sensitivity in certain applications. The polyclonal nature provides robust detection capabilities across various techniques, particularly in immunohistochemistry and immunofluorescence applications .
Applications of SRGAP1 Antibodies
SRGAP1 antibodies have been employed in numerous research applications, supporting investigations into cellular signaling, disease mechanisms, and developmental processes. Their versatility across multiple experimental platforms has made them invaluable tools in both basic and translational research.
Immunodetection Techniques
SRGAP1 antibodies are extensively used in various immunodetection methods:
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Western Blotting (WB): For quantitative assessment of SRGAP1 protein expression in cell and tissue lysates, enabling comparison between normal and pathological samples .
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Immunohistochemistry (IHC): For visualization of SRGAP1 expression patterns in tissue sections, particularly useful in cancer studies where SRGAP1 shows differential expression .
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Immunofluorescence (IF): For subcellular localization studies, allowing researchers to track SRGAP1 distribution in response to stimuli such as Slit2 treatment .
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Immunoprecipitation (IP): For isolation of SRGAP1 protein complexes, facilitating studies of protein-protein interactions, such as the interaction between SRGAP1 and Robo1 .
Research Applications
SRGAP1 antibodies have been instrumental in numerous research contexts:
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Cancer Research: Investigation of SRGAP1's role in gastric cancer, colorectal cancer, and other malignancies, where it functions as an oncogenic factor .
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Neurobiological Studies: Examination of SRGAP1's function in neuronal development, particularly in cell migration and axon guidance .
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Kidney Research: Studies of podocyte foot process regulation, where SRGAP1 controls small Rho GTPases .
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Cellular Junction Studies: Investigation of RhoA signaling modulation during epithelial junction maturation .
SRGAP1 Structure and Function
Understanding SRGAP1's structure and function is essential for interpreting research utilizing SRGAP1 antibodies. This knowledge provides context for antibody targeting and experimental design.
Protein Structure
SRGAP1 contains several conserved domains that contribute to its function:
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FCH (Fes/CIP4 homology) domain: Present at the N-terminus, contributes to membrane binding.
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Rho-GAP domain: Central regulatory domain responsible for GTPase-activating function.
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SH3 domain: C-terminal domain involved in protein-protein interactions .
This multi-domain structure enables SRGAP1 to interact with membrane components, regulate GTPase activity, and form protein complexes that mediate downstream signaling.
Cellular Functions
SRGAP1 plays pivotal roles in several cellular processes:
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Regulation of Rho GTPases: SRGAP1 enhances the intrinsic GTPase activity of Cdc42, promoting conversion to inactive GDP-bound form, which affects actin polymerization . Research has shown that SRGAP1 critically regulates RhoA, Cdc42, and Rac1 activity .
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Cell Migration and Invasion: SRGAP1 mediates the migration inhibition effect of Slit2-Robo1 signaling in colorectal cancer cells by inactivating Cdc42 .
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Cytoskeletal Reorganization: SRGAP1 influences F-actin distribution and organization, affecting cellular morphology and protrusion formation .
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Wnt/β-catenin Signaling: In gastric cancer, SRGAP1 activates the Wnt/β-catenin pathway, contributing to oncogenic processes .
Research Findings Utilizing SRGAP1 Antibodies
SRGAP1 antibodies have facilitated numerous significant research discoveries across multiple fields. The following subsections highlight key findings enabled by these antibodies.
SRGAP1 in Cancer Progression
Research using SRGAP1 antibodies has revealed significant insights into cancer biology:
Molecular Mechanisms Elucidated
SRGAP1 antibodies have helped uncover several molecular mechanisms:
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Slit2-Robo1 Signaling: Immunoprecipitation and immunofluorescence assays using SRGAP1 antibodies confirmed that SRGAP1 is a Robo1-interacting protein that exhibits similar dynamic subcellular distribution after Slit2 treatment in colorectal cancer cells .
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GTPase Regulation: Studies using SRGAP1 antibodies demonstrated that suppression of SRGAP1 affected the amount of active GTP-bound RhoA, Rac1, and Cdc42, thus altering cell morphology and inhibiting cell migration .
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EMT Modulation: Immunofluorescence analysis with SRGAP1 antibodies showed that SRGAP1 knockdown led to decreased expression of N-cadherin and Vimentin, suggesting a role in epithelial-mesenchymal transition .
SRGAP1 in Epithelial Junction Maturation
Research using SRGAP1 antibodies revealed its role in epithelial junctions:
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RhoA Signaling: Studies demonstrated that SRGAP1 is present at subconfluent junctions to a greater extent than in confluent cultures, and SRGAP1 RNAi restores RhoA signaling and contractility in subconfluent cultures to levels seen in confluent cells .
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Contractility Regulation: Laser ablation studies revealed higher recoil velocities in subconfluent SRGAP1 knockdown cells, similar to those seen in confluent conditions, suggesting SRGAP1's role in junctional tension development .
Antibody Validation and Quality Control
Manufacturers employ various validation methods to ensure SRGAP1 antibody specificity and reliability:
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Protein Array Testing: The specificity of some SRGAP1 antibodies is verified on a Protein Array containing the target protein plus 383 other non-specific proteins .
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Western Blot Validation: Antibodies are tested against cell lysates from multiple cell lines known to express or not express SRGAP1.
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Immunofluorescence Confirmation: Subcellular localization patterns are confirmed to match expected distribution patterns for SRGAP1.
Product Specs
Target Background
- Studies have shown that SRGAP1 protein expression is significantly reduced in 47.5% of colorectal cancer (CRC) tissues. This reduction is associated with tumor progression and poor prognosis. PMID: 27923383
- A consistently replicated locus at 12q14.2 (rs11175194 in SRGAP1, P(meta) = 1.14 x 10(-7)) has been identified. PMID: 25134534
- Mutations in the SLIT2-ROBO2 pathway genes, including SLIT2 and SRGAP1, are associated with an increased risk of congenital anomalies of the kidney and urinary tract. PMID: 26026792
- Elevated miR-145 levels in invasive glioblastoma cells (IM3 cells) target and down-regulate srGAP1, allowing downstream G-proteins to remain active and contribute to the observed invasive phenotype. PMID: 26026080
- The interaction of betaPix with srGAP1 is crucial for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration. PMID: 25150978
- Research indicates that srGAP1 exhibits GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. PMID: 24006490
- Genome-wide linkage analysis in populations in Ohio and Poland suggests that missense mutations in SRGAP1 are linked to genetic predisposition to papillary thyroid carcinoma. PMID: 23539728
- This study proposes that SRGAP1 expression in the anterior neocortex marks the early location of the human motor cortex, including its corticospinal projection neurons, enabling further investigation of their early differentiation. PMID: 21060114
- FNBP2, ARHGAP13, ARHGAP14 and ARHGAP4 constitute the FNBP2 family, characterized by FCH, RhoGAP and SH3 domains. PMID: 12736724
Q&A
What is SRGAP1 and what cellular functions does it regulate?
SRGAP1 is a GTPase-activating protein (GAP) that specifically regulates RhoA and Cdc42 small GTPases. It plays a crucial role in the signaling pathway that mediates the repulsive signaling of Robo and Slit proteins during neuronal migration. When SLIT2 interacts with ROBO1, it enhances the interaction between SRGAP1 and ROBO1, which subsequently leads to the inactivation of CDC42 . This mechanism is fundamental for proper neuronal migration and axon guidance during development. Understanding SRGAP1's function provides insights into neuronal development and potential neurological disorders related to axon guidance defects.
What are the typical applications for SRGAP1 antibodies in research?
SRGAP1 antibodies are validated for multiple experimental applications, including:
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Western Blotting (WB): For detecting SRGAP1 protein expression levels in cell and tissue lysates
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Immunoprecipitation (IP): For isolating SRGAP1 protein complexes from biological samples
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Immunohistochemistry (IHC): For visualizing SRGAP1 expression patterns in tissue sections
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Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of SRGAP1
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Immunofluorescence (IF): For subcellular localization studies
Different antibodies demonstrate varying performance across these applications, so researchers should select antibodies validated specifically for their intended experimental approach.
What is the expected molecular weight when detecting SRGAP1 in Western blot experiments?
| Observed Band Size | Possible Explanation |
|---|---|
| 150 kDa | Post-translational modifications such as phosphorylation or glycosylation |
| 124 kDa | Full-length unmodified protein |
| 80 kDa | Potential splice variant or proteolytic fragment |
| 75 kDa | Potential splice variant or proteolytic fragment |
When performing Western blot analysis, these variations should be considered when interpreting results . The presence of multiple bands may indicate different isoforms, degradation products, or post-translational modifications of the protein.
Which cell lines and tissues have been validated for SRGAP1 antibody reactivity?
SRGAP1 antibodies have been validated in various biological samples:
| Cell Lines | Tissues |
|---|---|
| HeLa cells | Human liver cancer tissue |
| HEK-293 cells | Human kidney tissue |
| SH-SY5Y cells | Mouse brain tissue |
| Apoptosised HeLa cells | Mouse kidney tissue |
| Mouse testis tissue |
These validated samples provide researchers with positive controls for experimental design and validation . Using these established cell lines and tissues as controls is recommended when establishing new experimental systems.
How should researchers optimize antigen retrieval for SRGAP1 immunohistochemistry?
Optimizing antigen retrieval is critical for successful SRGAP1 immunohistochemistry. Based on validated protocols:
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Primary recommendation: Use TE buffer (pH 9.0) for heat-induced epitope retrieval
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Alternative approach: Citrate buffer (pH 6.0) may be effective for certain tissue types
The optimal antigen retrieval method depends on tissue fixation conditions and the specific epitope recognized by the antibody. For formalin-fixed, paraffin-embedded (FFPE) tissues, a systematic comparison of both methods is recommended to determine optimal conditions for specific experimental contexts . Extended retrieval times may be necessary for heavily fixed tissues, and optimization should include testing different retrieval durations and temperatures.
How can researchers address potential cross-reactivity concerns with SRGAP1 antibodies?
When evaluating potential cross-reactivity of SRGAP1 antibodies, researchers should:
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Select antibodies targeting unique epitopes of SRGAP1 (different antibodies target distinct regions including AA 136-192, AA 469-497, AA 673-720, AA 952-1050, and full-length protein AA 1-1085)
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Include appropriate negative controls (knockout/knockdown samples where available)
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Validate specificity across multiple applications with independent antibodies recognizing different epitopes
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Consider potential homology with related proteins (particularly other SRGAP family members like SRGAP2 and SRGAP3)
Antibodies targeting the AA 952-1050 region appear to have high specificity for SRGAP1 across human and mouse samples, making them suitable for comparative studies across these species .
What are the optimal dilution ratios for different experimental applications of SRGAP1 antibodies?
Different applications require specific antibody dilutions for optimal signal-to-noise ratio:
| Application | Recommended Dilution Range | Notes |
|---|---|---|
| Western Blot (WB) | 1:1000-1:4000 | Lower dilutions may be needed for low-abundance samples |
| Immunoprecipitation (IP) | 0.5-4.0 μg per 1.0-3.0 mg total protein | Amount varies based on protein expression level |
| Immunohistochemistry (IHC) | 1:50-1:500 | Requires optimization for each tissue type |
| ELISA | Application-specific | Requires titration for each assay setup |
| Immunofluorescence (IF) | Application-specific | May require higher antibody concentrations than IHC |
These values provide starting points, but researchers should perform titration experiments for their specific samples to determine optimal conditions . Sample-dependent variables such as protein expression levels and sample preparation techniques may necessitate adjustments to these recommended dilutions.
How can researchers reconcile discrepancies in molecular weight observations for SRGAP1?
When investigating variations in observed molecular weight for SRGAP1 (predicted: 124 kDa vs. observed: 75-150 kDa), researchers should:
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Use protein extraction methods that preserve post-translational modifications (if studying the native form)
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Include phosphatase treatment controls to determine if higher molecular weight bands are due to phosphorylation
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Analyze tissue/cell-specific expression patterns as different isoforms may predominate in different systems
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Consider using isoform-specific antibodies targeting different domains of SRGAP1
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Incorporate protease inhibitors during sample preparation to prevent artificial degradation
The observation of multiple bands should be systematically investigated to determine whether they represent physiologically relevant isoforms or experimental artifacts . Correlation with mRNA expression data (e.g., RT-PCR for different splice variants) can help confirm the identity of observed protein variants.
What controls are essential when studying SRGAP1 in SLIT-ROBO signaling pathways?
When investigating SRGAP1's role in SLIT-ROBO signaling:
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Include positive controls: SH-SY5Y or HeLa cells with confirmed SRGAP1 expression
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Incorporate experimental manipulations of SLIT2 to observe changes in SRGAP1-ROBO1 interactions
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Use co-immunoprecipitation to confirm protein-protein interactions between SRGAP1, ROBO1, and CDC42
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Include appropriate knockdown/knockout controls to confirm antibody specificity
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Consider parallel analysis of other SRGAP family members to assess potential compensation mechanisms
Given SRGAP1's established role as a mediator in the SLIT2-ROBO1 signaling axis and its function in inactivating CDC42, experimental designs should account for these interactions when studying neuronal migration or axon guidance . Temporal analysis of these interactions during developmental processes can provide valuable insights into the dynamic regulation of this signaling pathway.
How should researchers approach SRGAP1 antibody validation for novel cell types or tissues?
When extending SRGAP1 antibody use to novel experimental systems:
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Begin with Western blot analysis to confirm expression and molecular weight in the new system
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Perform antibody titration to determine optimal concentration for each application
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Include positive controls from validated systems (e.g., HeLa cells, mouse brain tissue)
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Consider testing multiple antibodies targeting different epitopes of SRGAP1
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Validate findings with complementary approaches (e.g., mRNA expression, recombinant expression systems)
The cross-reactivity profile of each antibody should guide selection, with antibodies showing reactivity across human and mouse being valuable for comparative studies . For highly conserved regions, broader cross-species reactivity may be observed, though this should be experimentally verified.
What strategies can resolve contradictory results when using different SRGAP1 antibodies?
When faced with discrepant results using different SRGAP1 antibodies:
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Compare the epitope specificity of each antibody (consider potential isoform-specific recognition)
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Evaluate antibody performance across multiple applications to identify consistent patterns
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Incorporate genetic approaches (siRNA, CRISPR) to validate antibody specificity
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Consider native protein conformation effects on epitope accessibility in different applications
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Examine batch-to-batch variations by requesting validation data from manufacturers
Systematic comparison of monoclonal antibodies (e.g., clones 5D10 and 5D2) with polyclonal antibodies can help resolve discrepancies by leveraging their different recognition characteristics . Monoclonal antibodies offer higher specificity but may be sensitive to epitope modifications, while polyclonal antibodies provide more robust detection but potential higher background.
