SRT-Tag Monoclonal Antibody

What is the SRT-Tag and what specific sequence does it recognize?

The SRT-Tag (SRTag) is a 10-amino acid epitope (TFIGAIATDT) derived from the crystalline surface layer protein of Rickettsia typhi. This sequence is specifically recognized by the mouse monoclonal antibody SRT10, which was originally developed against this bacterial protein. The tag can be genetically fused to proteins of interest, enabling their detection and purification using the SRT10 antibody .

How does the SRT-Tag system compare to other epitope tag systems?

The SRT-Tag system offers several advantages similar to other epitope tag systems. Like common epitope tags (FLAG, HA, Myc), it enables protein detection without requiring antibodies against the protein of interest itself. The SRT-Tag has been successfully used for various applications including immunoblotting, immunocytochemistry, and immunoprecipitation. Its relatively small size (10 amino acids) minimizes potential interference with protein function, structure, or localization .

What detection methods are compatible with the SRT10 antibody?

The SRT10 monoclonal antibody has demonstrated effectiveness across multiple detection platforms. Research has confirmed its utility in:

• Immunoblotting (Western blotting) for protein detection in cell lysates

• Immunocytochemistry for visualizing protein localization in cells

• Immunoprecipitation for isolating protein complexes
  These methods have been validated with the SRT-Tag fused to various proteins including NCC27/CLIC1, MEF2D, and CD4 .

How is the SRT-Tag incorporated into recombinant proteins?

The SRT-Tag sequence can be incorporated into recombinant proteins through molecular cloning techniques. The oligonucleotide sequence encoding the SRTag epitope (TFIGAIATDT) is inserted into an expression vector containing multiple cloning sites. This allows the tag to be fused in-frame with the coding region of the gene of interest. For optimal implementation, researchers have successfully created mammalian expression vectors with the SRTag sequence, enabling the expression of fusion proteins with the tag positioned at either the N-terminus or C-terminus of the target protein .

What methodological considerations should be evaluated when SRT-Tag detection yields inconsistent results?

When encountering inconsistent detection results with the SRT-Tag system, researchers should systematically evaluate several factors:

• Epitope accessibility: The three-dimensional conformation of the fusion protein may obscure the SRTag epitope

• Expression levels: Low expression of the tagged protein might result in signals below detection threshold

• Antibody concentration optimization: Suboptimal primary or secondary antibody concentrations can affect signal-to-noise ratio

• Detection system sensitivity: Different visualization methods (chemiluminescence, fluorescence) have varying sensitivity thresholds

• Protein degradation: The tagged portion of the protein may be cleaved or degraded during sample processing

• Fixation effects: For immunocytochemistry, different fixation methods may differentially preserve the epitope structure

Troubleshooting should proceed systematically through these potential variables to identify the limiting factor in the experimental system.

How can SRT-Tag applications be optimized for challenging cellular compartments?

Optimization strategies for detecting SRT-tagged proteins in challenging cellular compartments (e.g., nucleus, mitochondria, membrane-bound organelles) include:

• Permeabilization protocol modification: Enhanced permeabilization may be required for detecting proteins in membrane-bound organelles

• Fixation method selection: Different fixation protocols (paraformaldehyde, methanol, acetone) may better preserve epitope accessibility in different cellular compartments

• Signal amplification: Using signal enhancement systems for low-abundance proteins

• Subcellular fractionation: Isolating specific cellular compartments prior to analysis

• Counterstaining with organelle markers: Confirming proper subcellular localization with compartment-specific markers

The successful detection of SRT-tagged proteins in different cellular locations (as demonstrated with nuclear MEF2D and membrane-associated CD4) suggests broad applicability across cellular compartments .

What are the optimal protocols for immunoprecipitation using the SRT10 antibody?

For immunoprecipitation using the SRT10 antibody, researchers should consider:

• Lysis buffer composition: Use buffers that preserve protein-protein interactions while effectively solubilizing the target protein

• Antibody-to-bead coupling: Optimal coupling of SRT10 to protein A/G beads or other immunoprecipitation matrices

• Binding conditions: Appropriate incubation time and temperature for maximizing antigen-antibody binding

• Washing stringency: Balance between removing non-specific binding while preserving specific interactions

• Elution methods: Consider both traditional elution under denaturing conditions and peptide-based elution using excess SRTag peptide for native protein recovery

The SRT10 antibody has been demonstrated to effectively immunoprecipitate SRT-tagged fusion proteins, confirming its utility in protein complex isolation .

How can the SRT-Tag system be implemented in protein purification strategies?

The SRT-Tag system can be effectively implemented in protein purification strategies through several approaches:

• Immunoaffinity chromatography: Immobilized SRT10 antibody columns can specifically capture SRT-tagged proteins

• Peptide elution: Competitive elution using synthetic SRTag peptide allows gentle, non-denaturing recovery of purified proteins

• Tandem purification: Combining SRT-Tag with other affinity tags (His, GST) for sequential purification steps to enhance purity

• Scale considerations: The system has been applied to both analytical and preparative scale purifications

Studies have demonstrated successful immunoaffinity purification of SRT-tagged human creatine kinase using peptide elution methods, confirming the utility of this approach for protein purification .

What optimization parameters are critical for immunocytochemistry with the SRT10 antibody?

Critical optimization parameters for immunocytochemistry using the SRT10 antibody include:

• Fixation method: Different fixation protocols (paraformaldehyde, methanol, acetone) may affect epitope accessibility

• Permeabilization conditions: Optimize detergent type and concentration for adequate antibody penetration while preserving cellular architecture

• Blocking conditions: Determine optimal blocking reagents to minimize background signal

• Antibody concentration: Titrate primary and secondary antibody concentrations for optimal signal-to-noise ratio

• Incubation parameters: Optimize time, temperature, and buffer composition for antibody binding

• Washing stringency: Balance between background reduction and signal preservation

• Detection system selection: Choose appropriate visualization method (fluorescence, chromogenic) based on experimental requirements

Successful immunocytochemical detection of SRT-tagged proteins including NCC27/CLIC1, MEF2D, and CD4 demonstrates the versatility of this system across different protein types and cellular localizations .

How has the SRT-Tag system been applied in protein-protein interaction studies?

The SRT-Tag system offers valuable approaches for protein-protein interaction studies:

• Co-immunoprecipitation: Using SRT10 antibody to pull down SRT-tagged proteins along with their interaction partners

• Proximity labeling: Combining SRT-Tag with enzyme-based proximity labeling techniques (BioID, APEX) to identify spatial protein relationships

• Interaction domain mapping: Creating truncated SRT-tagged protein variants to map interaction domains

• Competitive binding assays: Using excess SRTag peptide to disrupt/validate specific interactions

• Interactome analysis: Coupling SRT-Tag immunoprecipitation with mass spectrometry for unbiased interaction partner identification

The system's demonstrated compatibility with immunoprecipitation protocols makes it well-suited for these applications .

What are the considerations for using SRT-Tag in live cell imaging applications?

When adapting the SRT-Tag system for live cell imaging, researchers should consider:

• Antibody fragment engineering: Using Fab fragments or single-chain variable fragments (scFv) derived from SRT10 for improved cellular penetration

• Membrane permeability: Developing cell-permeable antibody derivatives or using microinjection/electroporation for intracellular delivery

• Fluorophore selection: Choosing appropriate fluorophores with minimal phototoxicity and photobleaching

• Expression timing: Optimizing the timing between protein expression and imaging to maximize signal while minimizing perturbation

• Alternative approaches: Considering recombinant expression of fluorescent protein-tagged anti-SRTag antibody fragments

While the current literature primarily describes fixed-cell applications, these considerations could guide adaptation for live cell imaging scenarios.

How can the SRT-Tag system be combined with other molecular biology techniques for advanced applications?

The SRT-Tag system can be integrated with various molecular biology techniques for advanced applications:

• Multiplexed detection: Combining SRT-Tag with other epitope tags (FLAG, HA, Myc) for simultaneous detection of multiple proteins

• CRISPR-Cas9 genome editing: Introducing the SRTag sequence into endogenous genes for tracking expression of native proteins

• Inducible expression systems: Coupling SRT-tagged proteins with tetracycline or other inducible promoters for temporal control

• Proteomics applications: Using SRT-Tag for affinity purification coupled with mass spectrometry (AP-MS)

• Structural biology: Employing SRT-Tag for protein purification prior to structural characterization by X-ray crystallography or cryo-EM

The demonstrated versatility of the SRT-Tag in different experimental contexts suggests its potential compatibility with these advanced applications.

What are the technical limitations of the SRT-Tag system that researchers should consider?

Researchers should consider several potential limitations when implementing the SRT-Tag system:

• Cross-reactivity: Possible recognition of endogenous proteins containing sequences similar to SRTag

• Expression system compatibility: Performance may vary across different expression systems (bacterial, yeast, insect, mammalian)

• Structural impact: Potential effects on protein folding, function, or interactions depending on tag placement

• Antibody production considerations: Dependency on continued availability of the SRT10 hybridoma or recombinant antibody production

• Detection sensitivity threshold: Lower limits of detection compared to enzymatic or fluorescent protein tags

Understanding these potential limitations allows researchers to design appropriate controls and validation experiments when implementing the SRT-Tag system.