Kallikrein-related peptidase 11 (KLK11), also known as hippostasin, is a serine protease involved in diverse physiological and pathological processes. It is implicated in cancer progression, chemoresistance, and metastasis, particularly in colorectal, ovarian, and breast cancers . KLK11 antibodies are critical tools for detecting and studying its expression in research and diagnostic settings.
KLK11 antibodies are produced in various formats, including polyclonal and monoclonal types, and are conjugated with labels such as biotin, fluorescent dyes (e.g., CL594), or enzymes for enhanced detection. While FITC (fluorescein isothiocyanate)-conjugated KLK11 antibodies are not explicitly listed in the provided sources, other conjugates are widely used for applications like flow cytometry, immunofluorescence (IF), and Western blotting (WB).
KLK11 antibodies are utilized in:
Flow Cytometry: CL594-67427 targets intracellular KLK11 in HepG2 cells .
Immunohistochemistry (IHC): Polyclonal antibodies detect KLK11 in prostate tissue .
Western Blotting (WB): Monoclonal antibodies recognize KLK11 isoforms (27–40 kDa) .
Typical FITC Applications (Hypothetical):
FITC-conjugated antibodies would enable direct fluorescence detection in:
Immunofluorescence: Visualizing KLK11 in cellular compartments.
Flow Cytometry: Quantifying KLK11 expression in cancer cells.
KLK11 overexpression correlates with oxaliplatin resistance in metastatic colorectal cancer (mCRC). Knockdown of KLK11 in resistant cell lines (e.g., HCT-8/L-OHP) reverses chemoresistance by suppressing the PI3K/AKT pathway, reducing proliferation and inducing apoptosis .
High KLK11 mRNA levels in TNBC tissues are associated with poor prognosis. KLK11 and KLK10 exhibit strong co-expression, suggesting coordinated roles in tumor progression .
Elevated serum KLK11 levels are observed in ovarian (70%) and prostate (60%) cancers, positioning it as a potential diagnostic biomarker .
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 .
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:
Cell Lines | Tissue Types |
---|---|
TT cells | Human breast cancer tissue |
DU 145 cells | Human prostate cancer tissue |
LNCaP cells | Pig brain tissue |
PC-3 cells | Rat brain tissue |
Jurkat cells | Mouse brain tissue |
A431 cells | Rat skin tissue |
HaCaT cells | |
HT-29 cells | |
SW 1990 cells | |
HCT-8 cells |
For optimal antibody validation, researchers should include these positive control samples in their experimental design .
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)
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:
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.
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 (++)
This scoring system has been validated in studies correlating KLK11 expression with clinicopathological features in metastatic colorectal cancer patients .
To effectively study KLK11's role in apoptotic pathways:
Use a combination of techniques to assess various apoptotic markers in relation to KLK11 expression:
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:
Research has demonstrated that KLK11 silencing increases L-OHP-induced apoptosis through activation of caspase-3 cleavage and modulation of the apoptosis signaling pathway .
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:
Biomarker validation:
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 .
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)
Research has elucidated several molecular mechanisms through which KLK11 mediates chemoresistance in colorectal cancer:
PI3K/AKT pathway activation:
Regulation of multi-drug resistance genes:
Modulation of apoptotic pathways:
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
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
Researchers may encounter several challenges when using KLK11 antibodies in Western blot:
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.