E-cadherin antibody (AA 375-631)

Catalog No. ABIN1858334

Product Details anti-E-cadherin Antibody

Target: E-cadherin (CDH1) (Cadherin 1, Type 1, E-Cadherin (Epithelial) (CDH1))

Binding Specificity: AA 375-631

Reactivity: Pig

Host: Rabbit

Clonality: Polyclonal

Conjugate: This E-cadherin antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Immunocytochemistry (ICC)

Purpose: Polyclonal Antibody to E-cadherin

Specificity: The antibody is a rabbit polyclonal antibody raised against E-cadherin. It has been selected for its ability to recognize E-cadherin in immunohistochemical staining and western blotting.

Purification: Antigen-specific affinity chromatography followed by Protein A affinity chromatography

Immunogen: Recombinant E-cadherin corresdonding to Phe375~Thr631 (Accession # C6EVT4) with N-terminal His and GST Tag

Isotype: IgG

Application Details

Application Notes:

Western blotting: 0.5-2 μg/mL

1:160-660 Immunohistochemistry: 5-20 μg/mL

1:16-66 Immunocytochemistry: 5-20 μg/mL

1:16-66 Optimal working dilutions must be determined by end user.

Comment:

The thermal stability is described by the loss rate. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37°C for 48h, and no obvious degradation and precipitation were observed. The loss rate is less than 5% within the expiration date under appropriate storage condition.

Restrictions: For Research Use only

Independent Validation (IF)

Validation #101345 (Immunofluorescence)

by: Sackler School of Biomedical Sciences, Tufts University School of Medicine

No.: #101345

Date: 06/05/2017

Antigen: E-cadherin

Lot Number: A20160427539

Method validated: Immunofluorescence

Positive Control: porcine unerupted tooth buds, dental epithelium

Negative Control: porcine unerupted tooth buds, mesenchym

Notes: Passed. The E-caherin antibody ABIN1858334 specifically labels epithelial tissues in porcine unerupted tooth buds in immunofluorescence, consistent with the expression pattern of E-cadherin.

Validation Images

A. ABIN1858334 stains the dental epithelium (DE, red) in pig tooth buds whereas the dental follicle (DF) and the pulp – labeled with the mesenchymal marker Vimentin (green) – remain unstained. B. Staining of the dental epithelial root tissue with ABIN1858334. C. Immunostaining of the DE with ABIN1858334. No staining of of dental mesenchym (DM) by ABIN1858334 or of the dental epithelium when using the secondary antibody alone (panle D). 40X, Scale Bar 50μm, 150 Resolution

Full Methods

Primary Antibody: ABIN1858334

Secondary Antibody: goat anti-rabbit AF568 conjugated antibody (ThermoFisher Scientific, A11011, lot 1778025)

Full Protocol:

• Harvest unerupted tooth buds from 5 month old pig jaws and wash 3x in phosphate bufferend saline (PBS) (Boston Bioproducts, BM-220).
• Fix tooth buds in 10% Formalin (Fisher Scientific, 23-245-685) for 24h.
• Wash 3x in PBS.
• Place tooth buds through graded ethanol seris for 6h at each concentration: 50%, 70%, 80%, 90%, 100%, 100%, 100%.
• Place tooth buds in 3x 2h in 100% xylene.
• Place in molten paraffin 2x (TissuePrep 2 Paraffin, Fisher, T555-2) 12h.
• Embed tooth buds in paraffin and cool on a 4°C cold plate for 1h.
• Let set ON at RT.
• Cut blocks into 6µm sections using a microtome (ThermoFisher Scientific, HM 355S).
• Use a tissue floating bath to mount sections onto microscope slides.
• Heat sections on a hot plate for 1h at 45°C for 1h then ON at 55°C.
• Deparaffinize slide 2x 5 min in 100% xylene.
• Rehydrate sections in graded ethanol series for 2min for each concentration: 100%, 95%, 70%, 50%.
• Place slide in H₂O for 5min.
• Transfer slide to a plastic holder filled with warmed 10mM Citric Acid Solution (made from sodium citrate monobasic; Sigma, 71497) pH6.0 and incubate in a steamer for 20min.
• Cool submerged slide on benchtop for 30min.
• Wash slide 3x 5min in PBS.
• Create a hydrophobic barrier on slide around sections with PAP-pen (Vector Labs, H-4000, lot 5138).
• Place slide in a humidified chamber and block with 5% BSA for 20min at 37°C.
• Gently remove BSA by tapping slide.
• Incubate sections with primary antibody
• • rabbit anti-pig E-cadherin antibody (antibodies-online, ABIN1858334, lot A20160408626) diluted to 1:40 in 1% BSA or
  • and mouse anti-pig Vimentin (Santa Cruz Biotechnology, SC-6260, lot G2814) diluted to 1:500 in 1% BSA for 1h at RT.
• A no primary antibody negative control was incubated in parallel in 1% BSA.
• Wash in slide 3x 5min in PBS.
• Incubate sections in secondary antibodies
• • goat anti-rabbit AF568 conjugated antibody (ThermoFisher Scientific, A11011, lot 1778025) diluted to 1:100 in 1% BSA and
  • goat anti-mouse AF488 conjugated antibody (ThermoFisher Scientific, A11001, lot 1752514 ) diluted to 1:100 in 1% BSA for 1h at RT.
• Wash in slide 3x 5min in PBS.
• Add approximately 10μL of Hard Set Mounting Medium with DAPI (Vector Labs, H-1500, lot ZC1221) for each section and mount cover slip.

Handling

Format: Liquid

Concentration: Lot specific

Buffer: PBS, pH 7.4, containing 0.02 % Sodium azide, 50 % glycerol.

Preservative: Sodium azide

Precaution of Use: WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: 4 °C,-20 °C

Storage Comment: Store at 4°C for frequent use. Stored at -20°C in a manual defrost freezer for two year without detectable loss of activity. Avoid repeated freeze-thaw cycles.

Expiry Date: 24 months

References for anti-E-cadherin antibody (ABIN1858334)

Smith, Zhang, Schiele, Khademhosseini, Kuo, Yelick: "Developing a biomimetic tooth bud model." in: Journal of tissue engineering and regenerative medicine, (2017) (PubMed).

Monteiro, Smith, Angstadt, Zhang, Khademhosseini, Yelick: "Dental cell sheet biomimetic tooth bud model." in: Biomaterials, Vol. 106, pp. 167-79, (2016) (PubMed).

Target Details for E-cadherin

Target: E-cadherin (CDH1) (Cadherin 1, Type 1, E-Cadherin (Epithelial) (CDH1))

Alternative Name: E-cadherin (CDH1 Products)

Background: CDHE, CD324, CDH1, Arc1, ECAD, CDH-E, E-CAD, LCAM, UVO, Cadherin 1 Type 1, Uvomorulin, E-Cadherin, Calcium-Dependent Adhesion Protein, Epithelial, Liver Cell Adhesion Molecule

Pathways: WNT Signaling, Sensory Perception of Sound, Cell-Cell Junction Organization, Tube Formation