Krt18 Antibody, FITC conjugated

What is KRT18 and why is it an important research target?

KRT18 (Keratin, Type I Cytoskeletal 18) is a 45 kDa intracellular protein that serves as a member of the intermediate filament subfamily prominently represented in epithelial tissues. It forms heteropolymeric filaments with Keratin 8, creating essential cytoskeletal structures in single-layered epithelia. KRT18 plays critical roles in maintaining cellular structural integrity, protecting cells from mechanical and non-mechanical stresses, and regulating signaling pathways involved in apoptosis.

The protein is particularly valuable as a research target because it serves as a specific marker for epithelial cell differentiation, making it essential for cancer research, developmental biology, and tissue regeneration studies. KRT18's expression patterns can help identify epithelial-derived tumors and monitor epithelial-to-mesenchymal transitions during cancer progression.

What detection methods are compatible with KRT18 antibody FITC conjugates?

KRT18 antibody FITC conjugates are optimized for several detection methodologies, with varying applications depending on your experimental requirements:

The FITC conjugation (excitation/emission: 499/515 nm) makes these antibodies compatible with standard 488 nm laser lines found in most flow cytometers and fluorescence microscopes, eliminating the need for secondary antibody incubation steps.

How should KRT18 antibody FITC conjugates be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody functionality and fluorophore integrity. FITC conjugated antibodies require specific handling:

• Store in the dark at 2-8°C to prevent photobleaching of the FITC fluorophore.

• Avoid repeated freeze-thaw cycles which can compromise antibody structure and fluorescence intensity.

• If long-term storage is necessary, aliquot the antibody to minimize freeze-thaw events.

• Do not expose to prolonged light, as FITC is particularly susceptible to photobleaching.

• Most preparations contain sodium azide (typically 0.09-0.15 mM) as a preservative, which should be noted when designing downstream applications.

Properly stored antibodies typically maintain activity for at least 12 months, though specific product documentation should be consulted for definitive shelf-life information.

What cell types or tissues most strongly express KRT18?

KRT18 expression is predominantly confined to epithelial tissues, with varying expression levels depending on tissue type and differentiation state:

For research applications, the epithelial carcinoma cell line A431 and human breast carcinoma cell line PMC-42 are frequently used as positive controls, as they were used as immunogens for many commercial KRT18 antibodies.

What are the optimal fixation and permeabilization protocols for intracellular KRT18 detection?

Since KRT18 is an intracellular cytoskeletal protein, proper fixation and permeabilization are critical for antibody access. The following protocol has been optimized for KRT18 detection while preserving epitope integrity:

• Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature.

• Wash cells 3× with PBS to remove excess fixative.

• Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes at room temperature.

• For flow cytometry applications, a methanol-based fixation/permeabilization (80% methanol, -20°C, 15 minutes) can preserve cytoskeletal structures effectively.

• Block with 5% BSA in PBS for 30-60 minutes prior to antibody incubation.

Avoid overfixation, which can mask epitopes and reduce signal intensity. For tissues with high keratin content, increasing permeabilization time may improve antibody penetration without compromising specificity.

How do different clones of KRT18 antibodies compare in epitope recognition and experimental performance?

Various monoclonal antibodies against KRT18 recognize distinct epitopes, affecting their utility in different applications:

When selecting between clones, consider that monoclonal antibodies offer higher specificity but may be sensitive to epitope masking during certain fixation protocols. Polyclonal antibodies typically provide stronger signals due to multiple epitope recognition but may exhibit higher background staining.

What are the considerations for multiplex staining when using KRT18 antibody FITC conjugates?

Multiplex staining requires careful planning to avoid spectral overlap and ensure proper compensation:

• FITC emission spectrum (peak: 515 nm) overlaps with PE and other green fluorophores, requiring proper compensation when designing multicolor panels.

• When using FITC-conjugated KRT18 antibodies with other fluorophores, select fluorochromes with minimal spectral overlap (e.g., APC, PE-Cy7).

• Always include single-stained controls for each fluorophore to establish compensation matrices.

• For tissue sections with high autofluorescence, consider using alternative conjugates with emission in far-red spectrum.

• Titrate antibodies when used in combination to minimize non-specific binding and optimize signal-to-noise ratios.

For optimal results in multiplex experiments, sequential staining may be preferable to simultaneous staining when using multiple intracellular markers to minimize antibody cross-reactivity.

How can I troubleshoot weak or non-specific signals in flow cytometry using KRT18 antibody FITC conjugates?

For optimal staining, 1-5 μg/ml antibody concentration is typically recommended for flow cytometry. If problems persist, testing different clone antibodies targeting different KRT18 epitopes may resolve detection issues.

What is the significance of KRT18 as a biomarker in cancer research and how can FITC-conjugated antibodies enhance detection?

KRT18 serves as a valuable biomarker in cancer research due to its specific expression pattern and release during cell death:

• Differential expression in carcinomas aids in tumor classification and identification of cells of epithelial origin.

• During apoptosis, caspase cleavage of KRT18 creates neo-epitopes that can be detected with specific antibodies.

• Circulating fragments of KRT18 (caspase-cleaved CK18) can serve as serum biomarkers for monitoring treatment response.

• Changes in KRT18 expression patterns correlate with epithelial-mesenchymal transition during cancer progression.

FITC-conjugated KRT18 antibodies enhance detection sensitivity in several ways:

• Direct conjugation eliminates secondary antibody steps, reducing background and improving signal-to-noise ratios.

• When used in flow cytometry, FITC conjugates enable quantitative assessment of KRT18 expression at the single-cell level.

• The bright fluorescence of FITC permits detection of low-abundance KRT18 in early-stage carcinomas or circulating tumor cells.

• In multiplexed assays, properly compensated FITC signals allow simultaneous assessment of multiple cancer markers.

How can I validate the specificity of a KRT18 antibody FITC conjugate?

Proper validation ensures experimental reliability and reproducibility:

• Positive controls: Use cell lines known to express high levels of KRT18, such as A431 or PMC-42 epithelial carcinoma cell lines.

• Negative controls: Include mesenchymal cell types or tissues that do not express KRT18.

• Blocking peptide controls: Pre-incubate antibody with the immunizing peptide to confirm binding specificity.

• Western blot validation: Confirm single band detection at approximately 45 kDa.

• Isotype controls: Use an isotype-matched, FITC-conjugated antibody (typically IgG1 for mouse monoclonals) to assess non-specific binding.

• Cross-reactivity testing: If working with non-human samples, validate performance in target species.

Robust validation protocols enhance confidence in experimental results and should be documented in publications to facilitate reproducibility.

What are the best control samples for experiments involving KRT18 antibody FITC conjugates?

Including these controls provides a comprehensive assessment of antibody performance and facilitates troubleshooting if unexpected results occur.

How do mouse monoclonal versus rabbit polyclonal KRT18 antibodies compare in terms of specificity and sensitivity?

For flow cytometry applications requiring high specificity, mouse monoclonal antibodies like clones C-04, DC-10, or N321 are frequently preferred. For applications where signal amplification is critical, rabbit polyclonal antibodies may offer advantages.

How can I optimize KRT18 antibody concentration for varying cell densities or tissue types?

Optimal antibody concentration varies with sample type, preparation method, and target expression level:

• For flow cytometry with cell lines expressing high KRT18 levels, 1-5 μg/ml is typically sufficient.

• For tissue sections or cells with lower expression, concentrations up to 10 μg/ml may be necessary.

• Titration experiment protocol:

  • Prepare serial dilutions of antibody (e.g., 10, 5, 2.5, 1.25, 0.625 μg/ml)

  • Stain identical aliquots of your sample with each concentration

  • Analyze signal-to-noise ratio for each concentration

  • Select the concentration that provides maximum specific signal with minimal background

When working with new sample types, always perform a titration experiment to determine optimal concentration, as excess antibody can increase non-specific binding while insufficient antibody reduces detection sensitivity.

What technical considerations should be made when using KRT18 antibody FITC conjugates for detecting apoptosis or cellular stress?

KRT18 undergoes specific modifications during apoptosis and cellular stress:

• During early apoptosis, caspases cleave KRT18 at specific sites, creating neo-epitopes detectable with specialized antibodies.

• For apoptosis studies, pair KRT18-FITC with annexin V or TUNEL assays for comprehensive assessment.

• Fixation protocols significantly impact epitope preservation – mild fixation (2% paraformaldehyde, 10 minutes) often preserves apoptotic epitopes better than harsh fixation.

• When detecting stress-induced KRT18 modifications:

  • Avoid detergents that may extract soluble KRT18 fragments

  • Process samples rapidly to prevent post-collection modifications

  • Include protease inhibitors in buffers to prevent artifactual degradation

For multiplex studies examining apoptosis, the FITC emission spectrum permits pairing with far-red fluorophores like APC for annexin V or PI for dead cell exclusion, minimizing compensation challenges.

How can I quantitatively assess KRT18 expression levels using flow cytometry?

Quantitative assessment of KRT18 expression provides valuable insights into epithelial differentiation and cancer progression:

• Standard mean fluorescence intensity (MFI) measurements:

  • Subtract isotype control MFI from sample MFI for background correction

  • Compare relative expression between experimental groups

  • Report as fold-change or absolute MFI values

• For more precise quantification:

  • Use quantitative flow cytometry beads with known FITC molecules

  • Create standard curves correlating fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Convert sample fluorescence to absolute protein expression levels

• Alternative metrics:

  • Percent positive cells above threshold defines population heterogeneity

  • Stain index (SI) = (MFI positive - MFI negative) / (2 × SD of negative population)

Standardizing acquisition parameters between experiments is critical for longitudinal studies comparing KRT18 expression across multiple timepoints or treatment conditions.

What are emerging applications for KRT18 antibody FITC conjugates in cancer research?

KRT18 antibodies are finding innovative applications in cancer research beyond traditional detection:

• Circulating tumor cell (CTC) identification:

  • FITC-conjugated KRT18 antibodies enable rapid identification of epithelial-derived CTCs in peripheral blood

  • When combined with EpCAM and other epithelial markers, provides higher CTC detection sensitivity

  • Flow cytometry applications allow isolation of viable CTCs for downstream molecular analysis

• Monitoring epithelial-to-mesenchymal transition (EMT):

  • Quantitative assessment of KRT18 downregulation during EMT processes

  • Enables time-course studies of cancer progression models

  • Permits sorting of cells at different EMT stages based on KRT18 expression levels

• Therapeutic response monitoring:

  • KRT18 fragments released during therapy-induced apoptosis serve as pharmacodynamic markers

  • Flow cytometric detection of intracellular KRT18 fragmentation patterns correlates with treatment efficacy

  • Combined with other markers, enables multiparametric assessment of heterogeneous response patterns

These emerging applications leverage the specificity of KRT18 detection to advance precision oncology research and therapeutic development strategies.

How can I design experiments to compare different clones of KRT18 antibodies for my specific research application?

A systematic approach to antibody comparison ensures selection of optimal reagents:

• Side-by-side performance evaluation:

  • Test multiple clones simultaneously on identical samples

  • Use consistent protocols, concentrations, and acquisition parameters

  • Include appropriate positive and negative controls

• Multidimensional assessment criteria:

  • Signal intensity: Compare mean/median fluorescence intensity

  • Signal-to-noise ratio: Assess specific signal versus background

  • Specificity: Evaluate staining pattern in mixed cell populations

  • Reproducibility: Test day-to-day and lot-to-lot consistency

• Application-specific testing:

  • For flow cytometry: Compare resolution of positive/negative populations

  • For immunofluorescence: Assess subcellular localization precision

  • For multiplexed applications: Evaluate performance in antibody cocktails

• Documentation and standardization:

  • Record detailed methods including clone, concentration, lot number

  • Standardize image acquisition settings or flow cytometer parameters

  • Archive raw data for future reference when changing antibody lots

This systematic approach ensures selection of the optimal KRT18 antibody clone for your specific experimental requirements and facilitates long-term experimental consistency.

What are the considerations for using KRT18 antibody FITC conjugates in high-throughput screening applications?

High-throughput screening with KRT18 antibodies requires specific optimization strategies:

• Automation compatibility:

  • Select antibody formulations without BSA or other proteins that may interfere with liquid handling systems

  • Evaluate stability under automated processing conditions

  • Test performance in reduced-volume protocols

• Signal optimization:

  • Determine minimum incubation times that maintain adequate signal-to-noise ratios

  • Assess signal stability over acquisition timeframes (particularly important for plate-based cytometry)

  • Optimize cell density for consistent fluorescence detection

• Quality control:

  • Include internal standard controls on each plate to normalize plate-to-plate variation

  • Implement automated quality metrics for signal intensity and specificity

  • Establish acceptance criteria for assay performance

• Data processing:

  • Develop automated gating strategies for flow cytometry applications

  • Standardize image analysis algorithms for high-content screening

  • Implement normalization methods to correct for systematic variations

When properly optimized, FITC-conjugated KRT18 antibodies can provide robust, reproducible results in high-throughput screening applications for drug discovery, genetic screens, or biomarker identification studies.