Claudin18.2 Antibody [L24N19]

Catalog No.: F3733

Application: Reactivity: Mouse, Rat, Human

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

使用情報

Dilution
IHC1:500

Application
IHC

Source
Rabbit Monoclonal Antibody

Reactivity
Mouse, Rat, Human

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

ポジティブコントロール	Mouse stomach tissue; Rat stomach tissue; Human lung tissue; Human stomach cancer tissue; Human colon cancer tissue; Human stomach tissue; Human stomach cancer tissue
ネガティブコントロール

プロトコール

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

生物学的記述

Specificity
Claudin18.2 Antibody [L24N19] detects endogenous levels of total Claudin18.2 protein.

タンパク質の局在
細胞接着、細胞膜、細胞内膜系、密着結合

Uniprot ID
P56856

Clone
L24N19

Synonym(s)
UNQ778/PRO1572; CLDN18; Claudin-18

Background
Claudin 18.2 (CLDN18.2) is a gastric-specific isoform of the claudin tight junction family, encoded by the CLDN18 gene on chromosome 3q22, and is essential for forming the gastric mucosal barrier through its four transmembrane domains and two extracellular loops (ECL1/ECL2), which mediate cis and trans claudin-claudin interactions via conserved electrostatic motifs. The short intracellular C-terminal PDZ-binding tail recruits ZO-1/2 through phosphorylation-sensitive residues that link the complex to the actin cytoskeleton, thereby enabling paracellular H⁺ and Na⁺ impermeability and maintaining epithelial polarity in differentiated stomach mucosa, where CLDN18.2 localizes to tight junction (TJ) strands. As part of its core barrier function, CLDN18.2 polymerizes into continuous TJ strands that selectively block proton leakage from gastric acid, with ECL2 residues W221, N242, and E248 forming a shallow groove that mediates homotypic interactions regulating ion conductance (Km ~10–50 mM for Na⁺) and preventing luminal contents from accessing basolateral compartments. The cytoplasmic tail coordinates with occludin and JAM-1 in a cholesterol-dependent supramolecular complex to dynamically remodel junctions under stress, with PKC/ERK-mediated phosphorylation transiently increasing permeability without loss of polarity. In tumorigenesis, aberrant exposure of CLDN18.2 epitopes due to polarity loss allows antibody access, triggering ADCC/CDC and blocking metastatic dissemination. CLDN18.2 is expressed in 40–60% of gastric and gastroesophageal junction (GEJ) cancers, where it drives proliferation and therapy resistance via YAP/IGF1R/AKT signaling.

Background

Claudin 18.2 (CLDN18.2) is a gastric-specific isoform of the claudin tight junction family, encoded by the CLDN18 gene on chromosome 3q22, and is essential for forming the gastric mucosal barrier through its four transmembrane domains and two extracellular loops (ECL1/ECL2), which mediate cis and trans claudin-claudin interactions via conserved electrostatic motifs. The short intracellular C-terminal PDZ-binding tail recruits ZO-1/2 through phosphorylation-sensitive residues that link the complex to the actin cytoskeleton, thereby enabling paracellular H⁺ and Na⁺ impermeability and maintaining epithelial polarity in differentiated stomach mucosa, where CLDN18.2 localizes to tight junction (TJ) strands. As part of its core barrier function, CLDN18.2 polymerizes into continuous TJ strands that selectively block proton leakage from gastric acid, with ECL2 residues W221, N242, and E248 forming a shallow groove that mediates homotypic interactions regulating ion conductance (Km ~10–50 mM for Na⁺) and preventing luminal contents from accessing basolateral compartments. The cytoplasmic tail coordinates with occludin and JAM-1 in a cholesterol-dependent supramolecular complex to dynamically remodel junctions under stress, with PKC/ERK-mediated phosphorylation transiently increasing permeability without loss of polarity. In tumorigenesis, aberrant exposure of CLDN18.2 epitopes due to polarity loss allows antibody access, triggering ADCC/CDC and blocking metastatic dissemination. CLDN18.2 is expressed in 40–60% of gastric and gastroesophageal junction (GEJ) cancers, where it drives proliferation and therapy resistance via YAP/IGF1R/AKT signaling.

References
[1] Poniewierska-Baran A, et al. Cells. 2025 Aug 19;14(16):1285. [2] Kyuno D, et al. Tissue Barriers. 2022 Jan 2;10(1):1967080.

PVDFメンブレン孔径参考表

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%