WNT2B antibody (AA 301-391)

Catalog No. ABIN675728

Product Details anti-WNT2B Antibody

Target: WNT2B (Wingless-Type MMTV Integration Site Family, Member 2B (WNT2B))

Binding Specificity: AA 301-391

Reactivity: Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This WNT2B antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Frozen Sections) (IHC (fro))

Cross-Reactivity: Mouse, Rat

Predicted Reactivity: Human,Dog,Pig,Horse,Chicken,Rabbit

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human WNT2B

Isotype: IgG

Application Details

Application Notes: WB 1:300-5000
ELISA 1:500-1000
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #029638 (Immunohistochemistry)

by: Immunohistochemistry Core, NYU Langone

No.: #029638

Date: 03/14/2014

Lot Number: 120522

Method validated: Immunohistochemistry

Positive Control: Human kidney

Negative Control: Human liver

Notes: Faint but distinguishable signal was detected in positive control sample and not in negative control sample.

Validation Images

Figure 1: Human kidney tissue stained with anti-WNT2 (brown) and counterstained with hematoxylin.

Figure 2: Human liver tissue stained with anti-WNT2 (brown) and counterstained with hematoxylin.

Figure 3: Human kidney tissue stained with isotype control antibody (brown) and counterstained with hematoxylin.

Figure 1: Human kidney tissue stained with secondary antibody only (brown) and counterstained with hematoxylin.

Full Methods

Primary Antibody:

• Antigen: Wingless-Type MMTV Integration Site Family, Member 2B (WNT2B) (N-Term)
• Catalog number: ABIN675728
• Lot number: 120522

Secondary Antibody:

• Antibody: Biotinylated goat anti-rabbit/anti-mouse (Kit)
• Lot number: D07640BA

Full Protocol:

• Immunohistochemistry was performed on a Ventana NEXes automated platform; instrument manufacturer specific reagents are italicized.
• 1. Slides were preheated in convection oven at 60°C for 30 min
• 2. Deparaffinization procedure: - 3 changes of Xylene, 5 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of 95% Ethanol, 3 min each - Rinsed in distilled water, 3 changes
• 3. Heat retrieval procedure - Slides retrieved in 10.0 mM Citrate, pH6.0 in a 1000W microwave oven (~100°C) for 15 min. - Slides were allowed to cool (in citrate) for 30 min. - Slides were washed x 3 in Distilled water
• 4. NEXes instrument procedure, iView DAB paraffin protocol (*abridged*): - Slide chamber warmed to 37°C
• 5. Slides rinsed with *reaction buffer* x3
• 6. *iView Inhibitor (H2O2)* applied and incubated for 4 min
• 7. Slides rinsed with *reaction buffer*
• 8. Antibody Application - Primary antibody diluted 1:250 in PBS (100 microliter applied/slide) - Ventana Isotype control applied neat - Slides Incubated overnight at room temperature (~12 hours ~25°C)
• 9. Slides rinsed with *reaction buffer* x3
• 10. *iView Biotinylated IgG* applied and incubated for 8 min
• 11. Slides rinsed with *reaction buffer*
• 14. *iView Streptavidin-Horseradish Peroxidase* applied and incubated for 8 min
• 15. Slides rinsed with *reaction buffer*
• 16. *iView DAB/H2O2* applied and incubated for 8 min
• 17. Slides rinsed with *reaction buffer*
• 18. *iView Copper* applied and incubated for 4 min
• 19. Slides rinsed with *reaction buffer*
• 20. Slides washed in Dawn Detergent/tap water
• 21. Counterstain Procedure - Hematoxylin (Leica 560 MX) 30 sec - Slides washed in tap water, 1 min - Decolorized (10% Acetic Acid in 70% ethanol), 1 min - Slides washed in tap water, 1 min - Bluing (Austin Clear Ammonia), 1 min - Slides washed in tap water, 1 min
• 22. Dehydration/coverslipping procedure: - 3 changes of 95% Ethanol, 3 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of Xylene, 5 min each - Mounted with Permount
• 23. Imaging: Leica SCN 400F Whole Slide Scanner with Digital Image Hub and Leica Slidepath software

Experimental Notes:

• Deviations from protocol/procedure supplied by manufacturer (attached).
• Step 1: Heated tissue 60°C for 30 minutes; manufacturer heats for 45 minutes.
• Step 2: No ethanol wash was performed during deparaffinization; manufacturer includes 1 wash of 80% ethanol for 3 minutes.
• Step 3.1: Slides were heated for 15 minutes; manufacturer provides a range of 15-20 minutes.
• Step 3.2: Slides were cooled for 30 minutes; manufacturer cools for 20 minutes.
• Step 4: Italicized reagents and incubation time are fixed instrument parameters.
• Step 5: Secondary species-specific serum block not used; manufacturer blocks with 5% normal goat serum for 2 hours.
• Step 8.1: Antibody diluted in PBS at 1:250; manufacture did not recommend diluent or dilution.
• Step 8.2.1: Primary antibody incubated at room temperature overnight; manufacturer incubates overnight 4°C with agitation.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

References for anti-WNT2B antibody (ABIN675728)

Li, Liu, Su, Ren, Zhu, Tian, Qiu: "MicroRNA-324-3p regulates nasopharyngeal carcinoma radioresistance by directly targeting WNT2B." in: European journal of cancer (Oxford, England : 1990), Vol. 49, Issue 11, pp. 2596-607, (2014) (PubMed).

Target Details for WNT2B

Target: WNT2B (Wingless-Type MMTV Integration Site Family, Member 2B (WNT2B))

Alternative Name: WNT2B (WNT2B Products)

Background:

Synonyms: WNT13, Protein Wnt-2b, Protein Wnt-13, WNT2B

Background: Ligand for members of the frizzled family of seven transmembrane receptors. Probable developmental protein. May be a signaling molecule which affects the development of discrete regions of tissues. Is likely to signal over only few cell diameters. May be involved in normal development or differentiation as well as in carcinogenesis.

Gene ID: 7482

UniProt: Q93097

Pathways: WNT Signaling