KLK11 Antibody, FITC conjugated

What is KLK11 and what cellular functions does it regulate?

KLK11 (Kallikrein-11) belongs to the peptidase S1 family and Kallikrein subfamily. It functions as a multifunctional protease that cleaves several substrates for kallikrein and trypsin and is involved in normal physiological processes in the bronchus . In pathological contexts, KLK11 has been implicated in cancer development, particularly colorectal cancer (CRC), where it appears to modulate cell proliferation and apoptosis pathways . Research methodologies for studying KLK11 function typically involve knockdown experiments using lentivirus-based shRNAs to evaluate changes in cell proliferation, apoptosis, and chemosensitivity .

Which tissue types and cell lines demonstrate reliable KLK11 expression for antibody validation?

KLK11 expression has been validated in numerous tissue types and cell lines. Based on immunohistochemical and Western blot analyses, positive KLK11 expression has been confirmed in:

For optimal antibody validation, researchers should include these positive control samples in their experimental design .

What are the recommended parameters for using KLK11 antibodies in immunofluorescence studies?

For immunofluorescence applications with FITC-conjugated KLK11 antibodies, the following methodological considerations should be implemented:

• Optimal dilution range: 1:200-1:800 for immunofluorescence on paraffin-embedded tissues (IF-P)

• Antigen retrieval: Use TE buffer pH 9.0; alternatively, citrate buffer pH 6.0 can be employed

• Blocking: Implement 5-10% normal serum from the same species as the secondary antibody

• Mounting medium: Use anti-fade mounting medium specifically designed for fluorescence to prevent photobleaching of the FITC conjugate

• Controls: Include positive control tissues (human breast cancer tissue has shown reliable results) and negative controls (omitting primary antibody)

How can KLK11 antibodies be utilized to investigate chemoresistance mechanisms in colorectal cancer?

KLK11 has been implicated in oxaliplatin (L-OHP) resistance in metastatic colorectal cancer (mCRC). A methodological approach to study this mechanism includes:

• Establishing resistant cell lines by exposing cells (e.g., HCT-8) to increasing concentrations of L-OHP

• Comparing KLK11 expression levels between parent and resistant cell lines using Western blot and RT-qPCR

• Implementing KLK11 knockdown in resistant cells followed by assessment of:

  • Cell viability using MTT assays

  • Apoptosis rates through flow cytometric analysis

  • Expression of multi-drug resistant genes (ABCC1, ABCB1, GSTP1, ERCC1)

  • PI3K/AKT pathway activation status

Research has demonstrated that KLK11 silencing reverses L-OHP resistance by inhibiting cell growth and activating apoptosis via suppression of the PI3K/AKT signaling pathway . FITC-conjugated antibodies can enable real-time visualization of KLK11 expression changes during development of resistance.

What scoring systems are recommended for quantifying KLK11 expression in immunohistochemical studies?

For standardized quantification of KLK11 expression in tissue samples, researchers should implement a dual-parameter scoring system that accounts for both staining intensity and percentage of positive cells:

Intensity Score:

• 0: No staining

• 1: Light yellow staining

• 2: Brown staining

• 3: Deep brown staining

Percentage of Positive Cells Score:

• 0: <5% stained cells

• 1: 5%-25% stained cells

• 2: 25%-50% stained cells

• 3: 50%-75% stained cells

• 4: >75% stained cells

The final score is calculated by multiplying these two values, with interpretation as follows:

• 0: Negative (-)

• 1-3: Weakly positive (+)

• 4-7: Moderately positive (++)

• 8-12: Strongly positive (+++)

This scoring system has been validated in studies correlating KLK11 expression with clinicopathological features in metastatic colorectal cancer patients .

How can researchers optimize detection of KLK11 in apoptotic pathway studies?

To effectively study KLK11's role in apoptotic pathways:

• Use a combination of techniques to assess various apoptotic markers in relation to KLK11 expression:

  • Western blotting to detect cleaved caspase-3 and Bcl-2/Bax ratio changes

  • Flow cytometry with Annexin V/PI dual staining to quantify apoptotic rates

  • RT-qPCR to measure mRNA expression of apoptosis-related genes

• When using FITC-conjugated KLK11 antibodies in flow cytometry, implement compensation controls to account for spectral overlap with other fluorophores like PI

• Methodological approach for establishing KLK11's role in apoptosis:

  • Conduct KLK11 knockdown experiments

  • Expose cells to apoptosis inducers (e.g., L-OHP at 5-10 μmol/l)

  • Measure changes in Bcl-2/Bax ratio and caspase-3 cleavage

  • Correlate these changes with KLK11 expression levels

Research has demonstrated that KLK11 silencing increases L-OHP-induced apoptosis through activation of caspase-3 cleavage and modulation of the apoptosis signaling pathway .

How can KLK11 antibodies be employed to evaluate its potential as a biomarker for Sjögren syndrome?

KLK11 has shown promise as a biomarker for Sjögren syndrome (SS). A methodological framework for investigating this application includes:

• Patient cohort selection: Include SS patients, dry eye disease (DED) patients, and normal controls (NL)

• Sample collection and processing:

  • Collect sera from study subjects

  • Use capture enzyme-linked immunosorbent assay (ELISA) to detect antibodies against KLK11

• Biomarker validation:

  • Calculate sensitivity and specificity using receiver operating characteristic (ROC) analysis

  • Determine optimal cutoff values for diagnostic use

Research has demonstrated significantly higher anti-KLK11 antibody levels in SS patients compared to both DED patients and normal controls. At an optical density cutoff point of 0.2695, anti-KLK11 antibody demonstrated 82% sensitivity and 94% specificity for distinguishing SS from other conditions .

What technical considerations should be addressed when using FITC-conjugated KLK11 antibodies in multi-parameter flow cytometry?

When implementing FITC-conjugated KLK11 antibodies in multi-parameter flow cytometry:

• Address spectral overlap:

  • FITC emits in the green spectrum (~519-525nm) and may overlap with other fluorophores

  • Perform proper compensation using single-stained controls

  • Consider the placement of KLK11-FITC in your panel based on expression level (bright markers are better placed in dimmer channels)

• Fixation and permeabilization:

  • Since KLK11 is primarily intracellular, use appropriate fixation and permeabilization protocols

  • Test different permeabilization reagents as they may affect FITC fluorescence intensity

• Titration of antibody:

  • Perform titration experiments to determine optimal concentration

  • Start with manufacturer's recommended dilution (typically 1:200-1:800 range)

  • Calculate signal-to-noise ratio at different concentrations to determine optimal staining

• Data analysis:

  • Use appropriate gating strategies to identify KLK11+ populations

  • Consider correlation with other markers of interest (e.g., apoptosis markers like Annexin V)

What are the molecular mechanisms through which KLK11 confers chemoresistance, and how can these be targeted?

Research has elucidated several molecular mechanisms through which KLK11 mediates chemoresistance in colorectal cancer:

• PI3K/AKT pathway activation:

  • KLK11 activates the PI3K/AKT signaling pathway, promoting cell survival and drug resistance

  • Knockdown of KLK11 suppresses this pathway, reversing resistance

• Regulation of multi-drug resistance genes:

  • KLK11 expression correlates with increased expression of ABCC1, ABCB1, GSTP1, and ERCC1

  • These genes are known mediators of drug efflux and DNA repair mechanisms

• Modulation of apoptotic pathways:

  • KLK11 expression decreases the Bcl-2/Bax ratio and inhibits caspase-3 cleavage

  • This creates an anti-apoptotic cellular environment conferring resistance to chemotherapy

Experimental approaches to target these mechanisms include:

• RNA interference targeting KLK11 (shRNA, siRNA)

• Small molecule inhibitors of PI3K/AKT pathway in combination with chemotherapy

• Evaluation of downstream apoptotic proteins as alternative targets

What contradictions exist in the literature regarding KLK11 function, and how can these be experimentally addressed?

Current literature presents several contradictions regarding KLK11 function that warrant further investigation:

Experimental approaches to address these contradictions include:

• Comparative analysis across tissue types:

  • Implement identical KLK11 knockdown experiments across diverse cell lines

  • Compare phenotypic outcomes (proliferation, apoptosis, migration) between tissue types

  • Use FITC-conjugated antibodies to quantify and visualize expression differences

• Autoimmunity investigation:

  • Develop animal models expressing KLK11 autoantibodies

  • Analyze immune cell populations and cytokine profiles

  • Explore molecular mimicry hypotheses through epitope mapping

• Functional domain analysis:

  • Use targeted mutagenesis to identify critical functional domains of KLK11

  • Correlate domain-specific mutations with phenotypic outcomes

What are common pitfalls when using KLK11 antibodies in Western blot applications, and how can they be addressed?

Researchers may encounter several challenges when using KLK11 antibodies in Western blot:

How should researchers design experiments to investigate potential interactions between KLK11 and other members of the kallikrein family?

For researchers investigating interactions between KLK11 and other kallikrein family members:

• Co-immunoprecipitation approach:

  • Use KLK11 antibody for pull-down experiments

  • Probe with antibodies against other kallikrein family members

  • Confirm specificity with reverse co-IP experiments

• Proximity ligation assay (PLA):

  • Utilize FITC-conjugated KLK11 antibody paired with antibodies against other kallikreins

  • Quantify interaction signals through fluorescence microscopy

  • Include appropriate controls (single antibody, non-related protein pairs)

• Functional interaction studies:

  • Implement simultaneous knockdown of KLK11 and other kallikreins

  • Compare phenotypic outcomes with single knockdowns

  • Analyze pathways where synergistic or antagonistic effects are observed

By implementing these methodological approaches, researchers can systematically investigate potential functional and physical interactions between KLK11 and other members of this important protease family.