Tmprss15 Antibody, FITC conjugated

What is Tmprss15 and why is it significant in research?

Tmprss15, also known as Enteropeptidase or Enterokinase, is a serine protease that plays a crucial role in digestive enzyme activation. It is responsible for initiating activation of pancreatic proteolytic proenzymes including trypsin, chymotrypsin, and carboxypeptidase A. The significance of this protein lies in its catalytic function of converting trypsinogen to trypsin, which subsequently activates other proenzymes including chymotrypsinogen, procarboxypeptidases, and proelastases . Research targeting Tmprss15 is valuable for understanding digestive enzyme cascades, pancreatic function, and potential therapeutic interventions for digestive disorders.

What is the optimal storage protocol for maintaining antibody activity?

For optimal maintenance of antibody activity, the FITC-conjugated Tmprss15 antibody should be stored at -20°C or -80°C upon receipt . The antibody formulation includes 50% glycerol and 0.03% Proclin 300 as a preservative in 0.01M PBS (pH 7.4) . This buffer composition helps maintain stability during storage. Critically, researchers should avoid repeated freeze-thaw cycles as these can significantly degrade antibody quality and performance . For projects requiring frequent use, consider aliquoting the antibody into single-use volumes before freezing to minimize freeze-thaw events.

How should researchers determine the optimal antibody dilution for their specific application?

Determining optimal antibody dilution requires systematic titration experiments customized to your specific application and sample type. Begin with a broad range of dilutions (e.g., 1:100, 1:500, 1:1000, 1:5000) in your experimental system. For ELISA applications with this particular antibody, analyze signal-to-noise ratios at each dilution by measuring specific binding to target versus background/non-specific binding. The optimal dilution provides maximum specific signal with minimal background. For fluorescence-based applications, additional factors to consider include signal intensity, photobleaching characteristics of FITC, and potential autofluorescence in your sample. Create a standardized protocol documenting the optimized conditions for reproducible results across experiments.

What controls are essential when using the FITC-conjugated Tmprss15 antibody?

A robust experimental design with appropriate controls is critical for reliable interpretation of results using this antibody. Essential controls include:

• Isotype control: Use a FITC-conjugated rabbit IgG (matching the host species and isotype of the Tmprss15 antibody) to assess non-specific binding .

• Negative tissue/cell control: Include samples known to lack Tmprss15 expression to evaluate background signals.

• Positive control: Incorporate samples with confirmed Tmprss15 expression (mouse tissue expressing enteropeptidase, particularly duodenal samples).

• Blocking peptide control: When available, pre-incubate the antibody with the immunogen peptide (recombinant Mouse Enteropeptidase protein aa 830-1069) to confirm binding specificity .

• Autofluorescence control: Examine unstained samples to assess natural fluorescence that might interfere with FITC signal detection.

What is the specific epitope recognized by this antibody and how does this influence experimental design?

This polyclonal antibody recognizes amino acids 830-1069 of mouse Tmprss15 (Enteropeptidase) . This region falls within the catalytic light chain of the protein after it undergoes cleavage into non-catalytic heavy chain and catalytic light chain components . The specificity for this region has important experimental implications:

• The antibody will detect both the full-length protein and the cleaved light chain.

• Experimental conditions that alter protein processing might affect detection.

• Cross-reactivity with human samples is not guaranteed due to species differences in this epitope region.

When designing experiments, researchers should consider the protein's post-translational modifications and processing state in their specific biological system, particularly if investigating proteolytic activation cascades.

What is the detailed composition of the antibody formulation and how might this affect experimental protocols?

The antibody is supplied as a liquid formulation with the following composition:

• Preservative: 0.03% Proclin 300

• Buffer constituents: 50% Glycerol, 0.01M PBS, pH 7.4

This formulation has several methodological implications:

• The high glycerol content (50%) requires accounting for dilution factors in applications requiring precise buffer conditions.

• Proclin 300 is compatible with most immunoassays but may affect certain enzymatic reactions or cell-based assays at higher concentrations.

• The neutral pH PBS buffer is generally compatible with most biological systems but may require adjustment for specialized applications.

For particularly sensitive applications, researchers might consider buffer exchange techniques if components of the formulation could potentially interfere with their experimental system.

How can researchers optimize dual immunolabeling protocols incorporating this FITC-conjugated antibody?

Optimizing dual immunolabeling with this FITC-conjugated Tmprss15 antibody requires careful consideration of fluorophore spectral properties and staining sequence. FITC has excitation/emission peaks at approximately 495/519 nm, so choose secondary fluorophores with minimal spectral overlap (e.g., Cy5, Texas Red). For dual staining protocols:

• Conduct preliminary single-staining controls to establish detection parameters for each antibody.

• Test different staining sequences to identify optimal order:

  • Simultaneous incubation of both primary antibodies

  • Sequential incubation with complete washing between steps

  • Applying FITC-conjugated antibody first or second

• Implement stringent blocking steps to minimize cross-reactivity between antibodies:

  • Use species-specific secondary antibodies when the second primary is from a different host

  • Include additional blocking steps between applications when using sequential protocols

• Adjust image acquisition settings to minimize bleed-through:

  • Capture single-labeled controls with identical settings

  • Employ sequential scanning for confocal microscopy

  • Consider linear unmixing algorithms for closely overlapping spectra

What approaches can address potential photobleaching issues when using this FITC-conjugated antibody in long-term imaging experiments?

FITC is relatively prone to photobleaching, which presents challenges for extended imaging sessions. Researchers can implement several strategies to mitigate this limitation:

• Chemical anti-fade reagents: Incorporate specialized mounting media containing anti-fade agents such as n-propyl gallate, DABCO, or commercial alternatives specifically designed for fluorescein preservation.

• Oxygen scavenging systems: Implement enzymatic oxygen scavenging systems (e.g., glucose oxidase/catalase with glucose) to reduce photobleaching rates during extended imaging.

• Acquisition parameters optimization:

  • Reduce excitation intensity to the minimum required for adequate signal detection

  • Minimize exposure times

  • Increase detector sensitivity (e.g., EM-CCD cameras, PMT voltage)

  • Use larger binning where resolution permits

• Advanced imaging techniques:

  • Implement time-lapse capture with intermittent imaging

  • Use deconvolution to extract more information from lower-intensity images

  • Consider computational approaches that correct for photobleaching effects

What strategies can address weak or absent signal when using this antibody in immunofluorescence applications?

When encountering weak or absent signal with the FITC-conjugated Tmprss15 antibody, systematically evaluate and optimize these key parameters:

• Epitope accessibility:

  • Test different fixation methods (paraformaldehyde, methanol, acetone)

  • Optimize permeabilization conditions (detergent type, concentration, duration)

  • Implement antigen retrieval methods (heat-induced, enzymatic)

• Antibody concentration and incubation conditions:

  • Test higher antibody concentrations

  • Extend incubation time (overnight at 4°C versus 1-2 hours at room temperature)

  • Optimize incubation buffer composition (BSA percentage, detergent concentration)

• Signal amplification techniques:

  • Consider tyramide signal amplification if compatible with experimental design

  • Use high-sensitivity detection systems or cameras

  • Implement image averaging during acquisition to improve signal-to-noise ratio

• Technical verification:

  • Confirm the functional integrity of the fluorophore using spectrophotometry

  • Verify target protein expression in your samples via alternative methods (Western blot, RT-PCR)

  • Test the antibody in a positive control sample with confirmed target expression

How can researchers differentiate between specific and non-specific binding when using this antibody?

Differentiating between specific and non-specific binding is crucial for accurate data interpretation. Implement these approaches to ensure signal specificity:

• Comprehensive controls implementation:

  • Compare staining patterns with isotype control at identical concentration

  • Include competitive blocking with the immunizing peptide (aa 830-1069 of mouse Tmprss15)

  • Include samples known to lack the target protein

  • Compare staining patterns with alternative antibodies targeting different epitopes of Tmprss15

• Signal pattern analysis:

  • Specific binding should show consistent subcellular localization corresponding to known biology

  • Non-specific binding often presents as diffuse, inconsistent, or unusual localization patterns

  • Compare observed patterns with published literature on Tmprss15 localization

• Quantitative assessment:

  • Calculate signal-to-background ratios under different conditions

  • Perform line-scan analysis across cellular regions to assess signal distribution

  • Compare fluorescence intensity between experimental and control samples

How does the sensitivity and specificity of this FITC-conjugated antibody compare to other detection methods for Tmprss15?

When evaluating this FITC-conjugated antibody against alternative Tmprss15 detection methods, consider these comparative factors:

What are the methodological advantages and limitations of using a FITC conjugate versus unconjugated primary antibodies?

Understanding the trade-offs between direct FITC conjugation and unconjugated antibodies allows researchers to select the optimal approach for their specific research questions:

Advantages of FITC-conjugated antibody:

• Simplified protocol with fewer steps, reducing hands-on time and potential for procedural errors

• Elimination of secondary antibody cross-reactivity concerns in multi-labeling experiments

• Direct 1:1 relationship between antibody and fluorophore, potentially improving signal consistency

• Reduced background in some applications due to elimination of secondary antibody binding to endogenous immunoglobulins

Limitations of FITC-conjugated antibody:

• Fixed fluorophore selection limits flexibility in imaging setup

• FITC is relatively prone to photobleaching compared to newer fluorophores

• No signal amplification from multiple secondary antibodies binding each primary

• Limited options for alternative detection methods (e.g., cannot easily switch to enzymatic detection)

For applications requiring maximum sensitivity or flexible detection options, unconjugated primary antibodies with separate detection reagents might be preferable. Conversely, for multicolor imaging or applications where protocol simplification is valuable, the FITC-conjugated format offers significant advantages.

How might post-translational modifications of Tmprss15 affect epitope recognition by this antibody?

The epitope recognized by this antibody (aa 830-1069 of mouse Tmprss15) may be subject to various post-translational modifications that could significantly impact detection. Researchers should consider these potential interactions:

• Proteolytic processing: Tmprss15/Enteropeptidase undergoes cleavage into heavy and light chains as part of its activation . Since the antibody targets a region within the light chain (catalytic domain), detection may be influenced by the protein's processing state.

• Glycosylation sites: The extracellular domain of Tmprss15 contains multiple N-glycosylation sites. While the exact impact on the epitope region requires experimental verification, glycosylation can potentially mask antibody binding sites or alter protein conformation.

• Phosphorylation: Serine proteases can be regulated by phosphorylation events. If phosphorylation sites exist within the epitope region, they could potentially enhance or inhibit antibody recognition.

To address these considerations, researchers might:

• Compare detection patterns in samples treated with glycosidases or phosphatases

• Analyze samples under reducing versus non-reducing conditions

• Compare detection in systems with altered protein processing machinery

What considerations are important when using this antibody for quantitative analysis of Tmprss15 expression levels?

For quantitative analysis of Tmprss15 expression using this FITC-conjugated antibody, researchers should implement these methodological considerations:

• Standardization protocols:

  • Establish standard curves using recombinant Tmprss15 protein at known concentrations

  • Include internal reference standards across all experimental batches

  • Maintain consistent imaging parameters between samples and across experiments

• Signal calibration approaches:

  • Use calibrated fluorescent beads to normalize fluorescence intensity measurements

  • Implement ratio-metric analysis against stable housekeeping proteins

  • Consider photobleaching rates in quantitative time-course experiments

• Technical validation:

  • Confirm linearity of detection within the expected expression range

  • Verify antibody saturation is not occurring at high target concentrations

  • Validate quantitative findings with orthogonal methods (e.g., Western blot, qPCR)

• Data analysis considerations:

  • Apply appropriate background subtraction methods

  • Use software that accounts for potential uneven illumination

  • Consider three-dimensional distribution of signal in confocal applications

How can this FITC-conjugated Tmprss15 antibody be integrated into multi-omics research approaches?

Integrating this antibody into multi-omics research requires thoughtful experimental design that leverages its specific capabilities while complementing other methodologies:

• Spatial proteomics integration:

  • Use the FITC-conjugated antibody to establish subcellular localization of Tmprss15

  • Correlate with mass spectrometry-based proteomics data on protein abundance

  • Combine with proximity labeling approaches to identify interaction partners

• Transcriptomics correlation:

  • Compare protein detection patterns with RNA-seq or single-cell RNA-seq data

  • Investigate potential post-transcriptional regulation by correlating mRNA and protein levels

  • Use the antibody to validate findings from transcriptomic screens in specific cell populations

• Functional genomics applications:

  • Apply the antibody to verify protein knockout/knockdown in CRISPR or RNAi experiments

  • Quantify protein expression changes following genetic manipulation

  • Correlate phenotypic outcomes with protein expression patterns

• Clinical research applications:

  • Develop standardized protocols for Tmprss15 detection in patient-derived samples

  • Correlate protein expression with clinical metadata and outcomes

  • Integrate with biomarker panels for digestive disorders

What emerging technologies might enhance the utility of this antibody in future research applications?

Emerging technologies offer opportunities to extend the applications and enhance the performance of this FITC-conjugated Tmprss15 antibody:

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize detailed subcellular localization beyond diffraction limits

  • Expansion microscopy to physically enlarge specimens for enhanced spatial resolution

  • Light-sheet microscopy for reduced photobleaching and rapid 3D imaging of large samples

• Microfluidic and single-cell applications:

  • Integration with microfluidic antibody capture techniques for ultra-sensitive detection

  • Adaptation for CyTOF/mass cytometry by metal-tagging for highly multiplexed analysis

  • Single-cell western blot technologies for correlative protein analysis at the cellular level

• Computational enhancements:

  • Machine learning algorithms for automated signal quantification and pattern recognition

  • Artificial intelligence-based image analysis to detect subtle expression differences

  • Cloud-based collaborative platforms for standardized antibody validation across laboratories