CLPS5 Antibody
Description
Claudin-5 (CLDN5) Antibodies
Claudin-5 is a critical tight junction protein in endothelial cells, and several well-characterized antibodies target its extracellular loop (ECR) or cytoplasmic domains. Key antibodies include:
PS5: A SOCS1-Derived Peptidomimetic
PS5 (UniProt ID: O00501) is a synthetic peptide mimicking the kinase inhibitory region (KIR) of Suppressor of Cytokine Signaling 1 (SOCS1). It is not an antibody but a therapeutic agent with antioxidant and anti-inflammatory properties:
Functional Characterization
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Clone 2B12: Shows high affinity (7.37 nM) for human CLDN5 and disrupts tight junctions in MDCKII cells, reducing trans-epithelial electrical resistance (TEER) by 60% .
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Clone 4F1: Binds both human and mouse CLDN5 but lacks functional inhibition .
Cross-Reactivity and Validation
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Engineered CLDN5 antigens (chimeric human/mouse ECR) induced antibodies in 85% of immunized mice .
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Antibodies like ab131259 show specificity for barrier proteins in IHC, with no cross-reactivity to CLDN1–4 or CLDN6–7 .
Table 1: Western Blot Performance of CLDN5 Antibodies
| Antibody | Observed MW (kDa) | Validated Tissues | Signal-to-Noise Ratio |
|---|---|---|---|
| 29767-1-AP | 22 | Lung (mouse/rat), Colon (human) | 12:1 |
| ab131259 | 20–25 | Lung squamous cell carcinoma | 15:1 |
Table 2: Immunohistochemistry Optimization
| Antigen Retrieval Buffer | Dilution | Staining Localization |
|---|---|---|
| Tris-EDTA (pH 9.0) | 1:1,000 | Cell membranes in colon |
| Citrate (pH 6.0) | 1:250 | Vascular endothelium in lung |
Limitations and Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
Basic Research Questions
What experimental applications are suitable for CLPS antibodies in pancreatic function studies?
CLPS (colipase) antibodies are primarily used to investigate lipid digestion mechanisms. Key applications include:
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Western blotting: Detect CLPS expression in pancreatic tissue lysates (30-50 µg/lane recommended) .
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Immunohistochemistry: Localize CLPS in acinar cells using formalin-fixed,paraffin-embedded sections .
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Co-factor activity assays: Validate CLPS-lipase interactions using in vitro lipid hydrolysis models .
Methodological Tip:
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Use reducing conditions (β-mercaptoethanol) for Western blotting, as CLPS has disulfide bonds critical for tertiary structure .
How can researchers validate CLPS antibody specificity in cross-species studies?
CLPS antibodies often exhibit cross-reactivity due to sequence homology:
| Species | Antigen Identity | Recommended Validation Test |
|---|---|---|
| Mouse | 73% | Knockout cell lysate control |
| Rat | 72% | Peptide blocking assay |
| Human | 100% | siRNA-mediated CLPS knockdown |
Data Contradiction Analysis:
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If unexpected bands appear in rodent models, perform mass spectrometry to confirm off-target binding .
Advanced Research Questions
What strategies mitigate interference from bile salts in CLPS functional assays?
Bile salts destabilize lipase-CLPS interactions, complicating in vitro studies. Solutions include:
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Micelle stabilization: Add 4 mM sodium taurodeoxycholate to reaction buffers.
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Kinetic assays: Monitor lipid hydrolysis at 37°C using pH-stat titration .
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Antibody pairing: Use CLPS antibodies with lipase-specific probes to track co-localization via fluorescence microscopy .
How do CLPS isoforms impact antibody performance in disease models?
Three CLPS isoforms (A, B, C) differ in N-terminal processing. Design considerations:
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Epitope mapping: Use antibodies targeting the conserved C-terminal domain (e.g., residues 80-100) .
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Isoform-specific assays: Combine Western blotting (distinguish molecular weights) and qPCR (isoform-specific primers).
| Isoform | Molecular Weight (kDa) | Tissue Specificity |
|---|---|---|
| A | 12.5 | Pancreas |
| B | 11.8 | Placenta |
| C | 10.9 | Fetal pancreas |
What controls are essential for CLPS antibody-based co-immunoprecipitation?
To study CLPS-lipase complexes:
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Negative control: IgG from pre-immune serum.
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Competition control: Add 10x molar excess of recombinant CLPS.
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Post-lysis stabilization: Include 1% n-dodecyl β-D-maltoside to preserve hydrophobic interactions .
