SRMS (UniProt ID: Q9H3Y6) is encoded by the SRMS gene (Gene ID: 6725) and regulates critical processes like keratinocyte proliferation and autophagy . Unlike the similarly abbreviated SRM (spermidine synthase), SRMS phosphorylates downstream targets such as DOK1 and modulates cancer cell growth .
Domains: Lacks myristylation and C-terminal regulatory sites, distinguishing it from other Src-family kinases .
SRMS antibodies are widely used in molecular research to study kinase activity, protein interactions, and therapeutic targeting.
Mechanism: SRMS inhibition via ibrutinib (FDA-approved kinase inhibitor) activates autophagy and suppresses tumor growth .
Experimental Model | Intervention | Outcome |
---|---|---|
U2OS cells (SRMS knockout) | Ibrutinib treatment | Reduced SRMS autophosphorylation |
SRMS(T302M) mutant | Ibrutinib exposure | No enzymatic inhibition |
Supplier | Catalog Number | Host | Applications | Reactivity | Price |
---|---|---|---|---|---|
Proteintech | 26447-1-AP | Rabbit | IP, IHC, ELISA | Human | $320+ |
Boster Bio | A06352-1 | Rabbit | IF, WB | Mouse | $370+ |
Novus Biologicals | NBP2-55947 | Rabbit | WB, IHC, ICC | Human, Mouse | $529+ |
SRMS (Tyrosine-protein kinase Srms) is a non-receptor tyrosine kinase involved in various cellular signaling pathways. Antibodies targeting SRMS are critical research tools that enable the detection, quantification, and characterization of this protein in biological systems. These antibodies facilitate investigations into SRMS expression patterns, localization, and functional roles in normal physiology and disease states. Most commercially available SRMS antibodies are rabbit polyclonal antibodies designed for research applications including Western blotting, immunohistochemistry, and immunofluorescence techniques . The importance of SRMS antibodies lies in their ability to provide insight into signaling pathways that may be relevant to various biological processes and potentially disease mechanisms.
SRMS antibodies have been validated for multiple experimental applications, with varying levels of reactivity depending on the specific antibody. Common applications include:
Western Blotting (WB): For detection of denatured SRMS protein in cell/tissue lysates
Immunohistochemistry (IHC/IHC-P): For localization of SRMS in tissue sections
Immunofluorescence (IF): For visualization of SRMS distribution in cells
When selecting an SRMS antibody, researchers should verify that the antibody has been validated specifically for their application of interest and target species. For example, some SRMS antibodies react with both human and mouse samples, while others are species-specific . Validation typically involves demonstrating specificity, sensitivity, and reproducibility for each application through standardized protocols.
Selecting the appropriate SRMS antibody requires consideration of several key factors:
Species Reactivity: Ensure the antibody recognizes SRMS in your experimental species (e.g., human, mouse, rat). Different antibodies have different cross-reactivity profiles .
Application Compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, ELISA). Some antibodies perform well in certain applications but not others .
Clonality: Determine whether a polyclonal or monoclonal antibody best suits your needs. Polyclonal antibodies (most common for SRMS) recognize multiple epitopes, potentially increasing sensitivity but with potential for cross-reactivity .
Validation Data: Review published validation data demonstrating the antibody's specificity and performance. Quality antibodies should have documentation showing absence of non-specific binding .
Binding Epitope: For specific research questions, consider antibodies targeting particular epitopes or domains of SRMS that may be relevant to your research hypothesis .
The selection process should involve thorough review of product documentation, published literature, and sometimes preliminary testing to ensure optimal performance in your experimental system.
Optimizing Western blotting with SRMS antibodies requires attention to several methodological considerations:
Sample Preparation:
Use fresh samples with appropriate protease inhibitors
Typical protein concentration: 20-50 μg total protein per lane
Denature samples in reducing buffer (with DTT or β-mercaptoethanol)
Electrophoresis and Transfer:
8-10% SDS-PAGE gels are typically suitable for SRMS (approximately 55 kDa)
Use standard PVDF or nitrocellulose membranes
Transfer at 100V for 60-90 minutes in cold transfer buffer
Antibody Incubation:
Blocking: 5% non-fat dry milk or BSA in TBST (1-2 hours at room temperature)
Primary antibody: Dilute SRMS antibody 1:500 to 1:2000 in blocking buffer
Incubation: Overnight at 4°C with gentle agitation
Secondary antibody: Anti-rabbit HRP-conjugated (1:5000-1:10000)
Detection and Optimization:
Use enhanced chemiluminescence (ECL) detection
Include positive and negative controls to validate specificity
If background is high, increase blocking time or adjust antibody dilution
For weak signals, extend exposure time or increase antibody concentration
These conditions may require optimization based on your specific experimental system and the particular SRMS antibody being used. Preliminary titration experiments are recommended to determine optimal antibody concentrations.
Optimizing immunohistochemistry (IHC) for SRMS detection requires methodical protocol development:
Tissue Preparation:
Fix tissues in 10% neutral buffered formalin (24-48 hours)
Paraffin embedding followed by sectioning at 4-6 μm thickness
Mount sections on positively charged slides
Antigen Retrieval (Critical Step):
Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)
Pressure cooker method: 121°C for 3-5 minutes or
Microwave method: 95-98°C for 15-20 minutes
Immunostaining Protocol:
Peroxidase blocking: 3% H₂O₂ in methanol (10 minutes)
Protein blocking: 5% normal goat serum (30-60 minutes)
Primary antibody: SRMS antibody diluted 1:100 to 1:500 in blocking buffer
Incubation: Overnight at 4°C or 1-2 hours at room temperature
Detection system: Polymer-based detection with DAB chromogen
Counterstain: Hematoxylin (light staining)
Validation and Controls:
Include positive control tissues known to express SRMS
Include negative controls (primary antibody omission)
Consider peptide competition assays to confirm specificity
Troubleshooting Common Issues:
Weak staining: Increase antibody concentration or incubation time
Non-specific background: Increase blocking time or dilute antibody
When implementing this protocol, systematic optimization of each step for your specific tissue type and fixation conditions will yield the most reliable results.
Several quantitative methods can be employed to accurately measure SRMS expression:
ELISA-Based Quantification:
Sandwich ELISA using anti-SRMS capture and detection antibodies
Typical sensitivity: 10-100 pg/mL
Standard curve generation using recombinant SRMS protein
Sample types: Cell lysates, tissue homogenates, serum/plasma
Mass Spectrometry-Based Quantification:
LC/ESI-SRM/MS (Liquid Chromatography/Electrospray Ionization-Selected Reaction Monitoring/Mass Spectrometry)
Selective quantification using unique peptides from SRMS
Internal standards: Stable isotope-labeled peptides or homologous peptides
Advantages: High specificity and multiplexing capability
Western Blot Densitometry:
Semi-quantitative assessment using image analysis software
Normalization to housekeeping proteins (β-actin, GAPDH)
Linear dynamic range: 10-fold to 20-fold concentration differences
Limitations: Lower precision than ELISA or MS methods
qRT-PCR for mRNA Expression:
Indirect measure of protein levels
Reference gene normalization (GAPDH, β-actin)
Correlation with protein levels should be validated
Each method offers distinct advantages, with ELISA providing high sensitivity for protein quantification, mass spectrometry offering superior specificity, and qRT-PCR enabling high-throughput analysis of transcript levels. Method selection should be guided by experimental requirements for sensitivity, specificity, and available sample material .
Integrating SRMS antibodies into Antibody Recruiting Molecule (ARM) strategies represents an advanced application at the intersection of immunology and chemical biology:
ARM Design Principles for SRMS Targeting:
ARMs are bifunctional molecules with two key components:
Implementation Strategies:
SRMS-Targeted ARMs:
Design TBM to bind specifically to cell-surface SRMS or associated proteins
ABM selection based on prevalent endogenous antibodies (anti-DNP, anti-αGal, anti-L-rhamnose)
Linker optimization for appropriate spatial arrangement and flexibility
Validation Approaches:
Flow cytometry to confirm ternary complex formation
Confocal microscopy for spatial arrangement visualization
Cytotoxicity assays to confirm immune-mediated clearance
Multivalency Considerations:
Potential Applications:
Targeted elimination of SRMS-overexpressing cancer cells
Immune redirection to SRMS-expressing pathological tissues
Development of novel immunotherapeutic approaches
This advanced application requires interdisciplinary expertise spanning antibody technology, medicinal chemistry, and immunology. Successful implementation depends on detailed characterization of SRMS expression patterns and careful design of ARM architecture to optimize the formation of ternary complexes between target cells, ARM molecules, and endogenous antibodies .
Addressing cross-reactivity concerns with SRMS antibodies requires rigorous validation strategies:
Cross-Reactivity Assessment Methods:
Knockout/Knockdown Validation:
CRISPR/Cas9-mediated SRMS knockout cell lines
siRNA knockdown of SRMS expression
Compare antibody reactivity between wild-type and knockout/knockdown samples
Orthogonal Detection Methods:
Mass spectrometry confirmation of detected protein bands
Parallel detection with multiple antibodies targeting different SRMS epitopes
Correlation of protein detection with mRNA expression data
Epitope Mapping and Competition Assays:
Peptide competition with the immunizing antigen
Alanine scanning mutagenesis to identify critical binding residues
Epitope mapping to identify potential cross-reactive regions
Homology Analysis:
In silico analysis of sequence similarity with related kinases
Pre-absorption with purified homologous proteins
Testing against panels of related tyrosine kinases
Cross-Reactivity Mitigation Strategies:
Antibody Purification:
Affinity purification against specific SRMS epitopes
Negative selection against potentially cross-reactive proteins
Epitope-specific purification for enhanced specificity
Assay Optimization:
Increased stringency in washing steps
Optimization of blocking reagents (specific to application)
Titration to determine optimal antibody concentration
Analytical Controls:
These methodologies provide a systematic approach to characterizing and addressing potential cross-reactivity, ensuring experimental results accurately reflect SRMS biology rather than artifacts from antibody non-specificity.
SRMS antibodies can be powerful tools for elucidating protein-protein interactions and signaling networks through several methodologies:
Co-Immunoprecipitation (Co-IP) Approaches:
Use SRMS antibodies to precipitate SRMS along with interacting partners
Mild lysis conditions to preserve protein complexes (1% NP-40 or CHAPS)
Reverse Co-IP with antibodies against suspected interaction partners
Mass spectrometry analysis of co-precipitated proteins for unbiased discovery
Controls: IgG control, SRMS-depleted lysates
Proximity Ligation Assay (PLA):
Detection of protein-protein interactions in situ with subcellular resolution
Requires SRMS antibody paired with antibody against potential interaction partner
Signal amplification allows detection of transient interactions
Quantitative assessment of interaction frequency in different cellular compartments
Phosphoproteomic Analysis:
Immunoprecipitation with SRMS antibodies followed by phosphopeptide enrichment
Identification of SRMS substrates via kinase assays with immunopurified SRMS
Phospho-specific antibodies to track SRMS-dependent phosphorylation events
Integration with inhibitor studies to establish causality in signaling cascades
FRET/BRET-Based Approaches:
Antibody-based FRET sensors for conformational changes in SRMS
Combination with genetically encoded fluorescent fusion proteins
Real-time monitoring of SRMS interactions and activation states
Spatial and temporal resolution of signaling events
Network Analysis Integration:
Correlation of SRMS interactome with transcriptomic changes
Pathway enrichment analysis of SRMS-associated proteins
Integration with publicly available protein interaction databases
Systems biology approaches to position SRMS within broader signaling networks
By employing these methodologies, researchers can systematically map the SRMS interactome and determine its functional role within cellular signaling networks. The combination of antibody-based isolation techniques with downstream analysis methods provides a powerful approach to understanding the complex biology of this tyrosine kinase .
Controlling experimental variability when working with SRMS antibodies requires systematic attention to multiple factors:
Sources of Variability and Mitigation Strategies:
Source of Variability | Manifestation | Control Measures |
---|---|---|
Antibody Lot-to-Lot Variation | Different signal intensity or background | - Validate each new lot against reference standard - Purchase larger lots for long-term studies - Maintain consistent antibody:protein ratio |
Sample Preparation Inconsistency | Variable protein recovery or degradation | - Standardize cell lysis procedures - Use fresh protease/phosphatase inhibitors - Control for post-translational modifications |
Technical Execution | Day-to-day variation in results | - Develop detailed SOPs - Maintain consistent incubation times/temperatures - Use automated systems where possible |
Biological Variation | Cell density, passage number effects | - Control cell confluence (70-80%) - Use consistent passage numbers - Pool biological replicates when appropriate |
Detection System Variability | Inconsistent signal development | - Standardize exposure times - Use internal loading controls - Employ digital imaging systems with linear range |
Quality Control Measures:
Reference Standards:
Maintain aliquots of positive control lysates/tissues
Include standard curve in quantitative assays
Use recombinant SRMS protein as positive control
Statistical Approaches:
Perform sufficient biological replicates (n≥3)
Apply appropriate statistical tests for experimental design
Consider power analysis for sample size determination
Documentation Practices:
Validating SRMS antibody specificity requires a multi-faceted approach tailored to your experimental system:
Comprehensive Validation Strategy:
Genetic Validation Approaches:
CRISPR/Cas9 knockout of SRMS gene
siRNA-mediated knockdown (transient)
Overexpression of tagged SRMS construct
Compare antibody reactivity patterns between manipulated and control samples
Biochemical Validation Methods:
Peptide competition assays with immunizing antigen
Pre-absorption with recombinant SRMS protein
Western blot analysis showing expected molecular weight band
Mass spectrometry confirmation of immunoprecipitated proteins
Orthogonal Detection Techniques:
Correlation between protein and mRNA levels
Comparative analysis with multiple SRMS antibodies targeting different epitopes
Immunoprecipitation followed by Western blot (IP-WB)
Immunofluorescence pattern correlation with GFP-tagged SRMS
Tissue/Cell Type-Specific Controls:
Tissues/cells known to express or lack SRMS
Comparison with established literature on expression patterns
Correlation with public database expression profiles
Developmental or stimulation-dependent expression changes
Documentation of Validation:
Maintain detailed records of all validation experiments
Document controls and experimental conditions
Establish accepted criteria for passing validation
Consider pre-registering validation protocols for transparency
Resolving contradictory results obtained with different SRMS antibodies requires systematic investigation and reconciliation strategies:
Analytical Framework for Resolving Contradictions:
Epitope-Based Analysis:
Map the epitopes recognized by each antibody
Consider whether different isoforms or post-translational modifications might affect epitope accessibility
Evaluate potential for conformational versus linear epitope recognition
Design experiments targeting conserved versus variable regions
Technical Reconciliation:
Standardize experimental conditions across antibodies
Optimize protocols individually for each antibody
Perform side-by-side comparisons under identical conditions
Consider whether different applications (WB vs. IHC vs. IF) yield consistent results
Orthogonal Validation Approaches:
Mass spectrometry analysis to confirm protein identity
Correlation with mRNA expression data
Genetic manipulation (overexpression, knockdown) to confirm specificity
Multiple detection methods with the same sample set
Structured Evaluation Protocol:
Develop a decision matrix for weighing evidence from different antibodies
Consider antibody generation methods (polyclonal vs. monoclonal)
Evaluate validation data provided by manufacturers
Consult literature for previous reconciliation of similar contradictions
Case-Based Resolution Strategies:
Contradiction Type | Investigation Approach | Resolution Strategy |
---|---|---|
Differential Localization | Co-staining with organelle markers | Determine if differences represent distinct pools or artifacts |
Expression Level Discrepancies | Titration experiments with recombinant standards | Establish relative sensitivity and linear range for each antibody |
Different MW Bands | Phosphatase treatment, isoform-specific PCR | Identify if bands represent modified forms or non-specific binding |
Stimulus-Response Differences | Time-course and dose-response with multiple antibodies | Determine temporal dynamics and sensitivity thresholds |
By systematically investigating the source of contradictions and integrating multiple lines of evidence, researchers can develop a more complete understanding of SRMS biology that accommodates or explains apparent contradictions in antibody-based experimental results .
Implementing SRMS antibodies in multiplexed detection systems enables simultaneous analysis of multiple targets:
Multiplexed Detection Methodologies:
Multiplex Immunofluorescence/Immunohistochemistry:
Sequential staining with different primary antibodies including anti-SRMS
Use of spectrally distinct fluorophores or chromogens
Tyramide signal amplification for increased sensitivity
Computational unmixing of spectral overlap
Applications: Tissue microenvironment analysis, signaling pathway activation
Mass Cytometry (CyTOF):
Metal-conjugated SRMS antibodies (typically lanthanides)
Simultaneous detection of 40+ cellular markers
Single-cell resolution with minimal spillover
Integration with cell surface markers and functional readouts
Applications: Heterogeneity analysis in complex cell populations
Proximity-Based Multiplexed Detection:
Antibody-oligonucleotide conjugates for proximity extension assays
Proximity ligation assays for protein-protein interactions involving SRMS
Digital counting of molecular interactions
Applications: Protein interaction networks, pathway activation status
Multiplex Western Blotting:
Size-based separation followed by multiple antibody detection
Fluorescent secondary antibodies with distinct spectra
Sequential stripping and reprobing with SRMS and other antibodies
Applications: Signaling pathway analysis, post-translational modifications
Optimization Strategies for Multiplexed Detection:
Antibody Selection Criteria:
Species compatibility (avoid same-species primaries)
Cross-reactivity testing against all targets in the panel
Validation in multiplex settings (not just individually)
Technical Considerations:
Optimized antibody concentration for each target
Order of antibody application in sequential staining
Appropriate blocking between rounds of staining
Data normalization and analysis workflows
Quality Control for Multiplexed Assays:
Multiplexed detection with SRMS antibodies provides contextual information about SRMS expression, activation, and function in relation to other cellular components, enabling systems-level analysis of its biological roles.
Using SRMS antibodies in therapeutic development research involves several specialized considerations:
Therapeutic Development Applications:
Target Validation Studies:
Correlation of SRMS expression with disease progression
Tissue-specific expression profiling using validated antibodies
Knockdown/knockout phenotype characterization
Patient stratification based on SRMS expression or activity
Mechanism of Action Studies:
Monitoring SRMS levels/activity during drug treatment
Pathway modulation assessment through phosphorylation status
Combination therapy effects on SRMS signaling networks
Resistance mechanism exploration in non-responding models
Biomarker Development:
Identification of SRMS as predictive/prognostic biomarker
Standardization of detection protocols for clinical application
Development of companion diagnostic assays
Correlation with other established biomarkers
Antibody-Drug Conjugate (ADC) Development:
Assessment of SRMS internalization dynamics
Epitope selection for optimal ADC delivery
Cytotoxic payload delivery efficiency studies
Off-target effects evaluation
Key Technical Considerations:
Regulatory and Standardization Requirements:
Clinical Laboratory Improvement Amendments (CLIA) validation
Reproducibility across different laboratories
Standardized protocols with defined cut-off values
Reference standard development and implementation
Clinical Sample Considerations:
Optimization for formalin-fixed paraffin-embedded (FFPE) tissues
Preanalytical variable control (fixation time, processing)
Decalcification effects on epitope preservation
Automated staining platform validation
Translation to Precision Medicine:
The application of SRMS antibodies in therapeutic development requires rigorous validation beyond research use, with particular attention to reproducibility, standardization, and clinical relevance of detection methods.
Emerging antibody technologies offer exciting possibilities for advancing SRMS research:
Next-Generation Antibody Technologies:
Recombinant Antibody Platforms:
Single-chain variable fragments (scFvs) targeting SRMS
Nanobodies (VHH) with enhanced tissue penetration
Bispecific antibodies targeting SRMS and effector cells/molecules
Benefits: Reduced batch-to-batch variation, defined specificity, renewable source
Spatially-Resolved Antibody-Based Detection:
Highly multiplexed imaging using DNA-barcoded antibodies
In situ sequencing with antibody-oligonucleotide conjugates
Spatial transcriptomics integrated with protein detection
Applications: Tissue microenvironment, subcellular localization, multi-omic integration
Dynamic Antibody-Based Biosensors:
FRET-based sensors using SRMS antibody fragments
Split-protein complementation assays for activity monitoring
Optogenetic integration for spatiotemporal control
Applications: Real-time monitoring of SRMS activity in living systems
AI-Enhanced Antibody Development:
Machine learning for epitope prediction and antibody design
Computational modeling of antibody-antigen interactions
Automated validation pipelines for high-throughput screening
Benefits: Reduced development time, enhanced specificity, optimized properties
Future Research Directions:
Single-Cell Analysis of SRMS Biology:
Integration with single-cell RNA sequencing
Mass cytometry for protein-level analysis
Spatial proteomics in tissue contexts
Applications: Cell heterogeneity, rare cell populations, microenvironment interactions
In Vivo Antibody-Based Imaging:
PET imaging with radiolabeled SRMS antibodies
Intravital microscopy with fluorescent antibody derivatives
Photoacoustic imaging for deep tissue visualization
Applications: Disease progression monitoring, therapeutic response assessment
Therapeutic Applications Building on ARM Technologies:
The integration of these emerging technologies with established SRMS antibody applications will likely accelerate discovery in areas including cancer biology, developmental processes, and immune regulation, while enabling new therapeutic approaches targeting SRMS-dependent pathways.
Ensuring reproducibility in SRMS antibody research requires attention to several critical factors throughout the experimental process:
Critical Reproducibility Considerations:
Antibody Selection and Documentation:
Document complete antibody information (manufacturer, catalog number, lot number)
Perform independent validation regardless of manufacturer claims
Consider using recombinant antibodies for improved consistency
Maintain detailed records of antibody performance across applications
Experimental Design Practices:
Include appropriate positive and negative controls
Perform biological replicates (n≥3) and technical replicates
Blind analysis where possible to minimize bias
Pre-register experimental protocols when feasible
Standardized Protocols:
Develop detailed standard operating procedures (SOPs)
Control for variables including sample preparation, incubation conditions, and detection parameters
Calibrate instruments regularly and document settings
Implement quality control checkpoints throughout protocols
Data Analysis and Reporting:
Use appropriate statistical methods with justification
Report all data points, not just representative images
Provide access to raw data when possible
Include detailed methodological information in publications
Cross-Laboratory Validation:
By systematically addressing these considerations, researchers can enhance the reliability and reproducibility of SRMS antibody-based experiments, ultimately accelerating scientific progress through increased confidence in published findings and reduced resource expenditure on irreproducible results.
SRMS antibody research offers significant potential for advancing disease mechanisms understanding and therapeutic development:
Disease Mechanism Insights:
Signaling Pathway Elucidation:
Identification of SRMS-dependent phosphorylation events
Mapping of SRMS interactome in normal versus disease states
Temporal dynamics of SRMS activation during disease progression
Integration with other tyrosine kinase signaling networks
Expression Pattern Analysis:
Tissue-specific and cell type-specific SRMS distribution
Changes in SRMS expression during disease development
Correlation with clinical outcomes and disease subtypes
Single-cell resolution of heterogeneous expression patterns
Functional Studies:
SRMS inhibition or activation effects on cellular phenotypes
Genetic-pharmacologic correlation studies
Compensation mechanisms in SRMS-targeted interventions
Biomarker identification for disease stratification
Therapeutic Opportunity Development:
Target Validation:
Confirmation of SRMS as druggable target
Identification of critical domains for functional inhibition
Patient stratification markers for precision medicine
Mechanism-based combination therapy rationales
Advanced Therapeutic Approaches:
Antibody-drug conjugates targeting SRMS-expressing cells
Antibody recruiting molecules (ARMs) for immune-mediated responses
Proteolysis targeting chimeras (PROTACs) for SRMS degradation
Bifunctional degraders linking SRMS to E3 ligases
Companion Diagnostics:
SRMS antibody research thus provides a foundation for translational advances by connecting molecular mechanisms to potential therapeutic interventions, enabling both deeper understanding of disease biology and more rational development of targeted treatments for conditions where SRMS plays a significant role.