Plakophilin 2 antibody (AA 801-881)

Catalog No. ABIN1386389

Product Details anti-Plakophilin 2 Antibody

Target: Plakophilin 2 (PKP2)

Binding Specificity: AA 801-881

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This Plakophilin 2 antibody is un-conjugated

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

Cross-Reactivity: Human

Predicted Reactivity: Mouse,Rat,Dog,Cow,Sheep,Pig,Horse,Rabbit

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human Plakophilin 2

Isotype: IgG

Application Details

Application Notes: 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
ICC 1:100-500

Restrictions: For Research Use only

Independent Validation (IF)

Validation #103656 (Immunofluorescence)

by: Gencardio (IDIBGI)

No.: #103656

Date: 03/13/2019

Antigen: PKP2

Lot Number: AD102900

Method validated: Immunofluorescence

Positive Control:

HL1 cells

HeLa cells

mouse heart tissue

Negative Control:

HL1 cells

HeLa cells

secondary antibody only control

Notes:

ABIN1386389 specifically labels the targeted antigen in mouse heart. No signal was detected in sample negative control and the secondary antibody only control.

Validation Images

A. IF staining of cells expressing PKP2-FLAG using ABIN1386389 (green) or an anti-FLAG-tag antibody (red). Nuclear staining with DAPI (blue) (magnification 60x). B. Staining of HeLa cells not expressing PKP2-FLAG using ABIN1386389 (magnification 40x). C. Staining of cells expressing PKP2-Flag with a secondary antibody only (magnification 40x). D and E. Staining of endogenous Pkp2 in mouse heart tissue with ABIN1386389 (D) or secondary antibody only (E) (magnification 40x).

Full Methods

Primary Antibody: ABIN1386389

Secondary Antibody: goat anti-rabbit AF488 antibody (Invitrogen, A11008, 1911238)

Full Protocol:

• HL1 and HeLa cells
• • Grow HL1 cells in Claycomb medium (sigma, 51800C, SLBX8700) supplemented with 10% FBS (Gibco, 10270-106, 42G5178K), 1% Penicillin-Streptomycin (Sigma, P4333, lot 048M4874V) and 1% Glutamax (Gibco, 35050-061, lot 2037045), at 37°C and 5% CO₂ in 12mm coverslips in a well of 12 well plate.
  • Grow HeLa cells in MEM/EBSS medium (GE Life Science, SH30244.01, lot AC14563283) supplemented with 10% FBS (Gibco, 10270-106, lot 42G5178K), 1% Penicillin-Streptomycin (Sigma, P4333, lot 048M4874V) and 1% Glutamax (Gibco, 35050-061, lot 2037045), at 37°C and 5% CO₂ in 12mm coverslips in a well of 12 well plate.
  • Transfect HeLa cells with PKP2a plasmid (p915 PKP2a-pFLAG-cmv5a, provided by Dr. Green KJ) using Lipofectamine 3000 (Invitrogen, L3000-015, lot 2041107) following the manufacturer´s instructions.
  • Wash cells 1x with PBS.
  • Fix cells on coverslips in 4% PFA for 20min at RT.
  • Wash cells 3x for 5min with PBS.
  • Incubate coverslips with quench solution (PBS with 75mM glycine and 20mM NH₄Cl) for 10min at RT.
  • Wash cells 1x with PBS.
  • Permeabilize cells and block non-specific binding with PFS-like buffer containing 0.1% BSA, 0.1% triton and 0.02% NaN₃ for 30min at 37°C.
  • Incubate cells with primary anti-Plakophilin 2 (PKP2) (AA 831-881) antibody (antibodies-online, ABIN1386389, lot AD102900) diluted 1:60 in PFS-like buffer or with anti-Flag-tag antibody (Sigma, monoclonal anti-flag, F316, lot SLBT7654) diluted 1:400 in PFS-like buffer ON at 4°C.
  • Wash cells 3x for 5min with PBS.
  • Incubate cells with secondary goat anti-rabbit AF488 antibody (Invitrogen, A11008, lot 1911238) or with secondary goat anti-mouse AF568 antibody (Invitrogen, A11031, lot 1841757) diluted 1:200 in PFS-like buffer for 30min at RT.
  • Wash cells 2x for 5min with PFS-like buffer.
  • Wash cells 2x for 5min with PBS.
  • DAPI counterstain for 5min at RT.
  • Mount coverslips on glass slides in Calbicochem (Millipore, 345789, lot 2682585).
  • Image acquisition with NIS-ELEMENTS (Nikon).
• Tissue: mouse heart
• • Obtain mouse heart mouse and wash it with PBS.
  • Fix tissue in 15ml falcon tube with 4% PFA ON at 4°C.
  • Wash the fixed heart with water.
  • Embed tissue in paraffin.
  • Cut the tissue using a microtrom and put it into slides.
  • Desparaffinize the tissue in the slide (from xylene to water).
  • Incubate the slides with retrieval solution for 30min at 95°C to permeabilize the tissue.
  • Cool slides down on ice.
  • Wash tissue 2x for 3min with PBS.
  • Wash tissue 2x for 3min with PBS 0.1% triton.
  • Block non-specific binding with PBS 0.1% triton 10% BSA for 1h at RT.
  • Incubate tissue with primary anti-PKP2 (AA 831-881) antibody (antibodies-online, ABIN1386389, lot AD102900) diluted 1:100 in PBS 1% BSA solution ON at 4°C.
  • Wash tissue 2x for 5min with PBS 0.1% triton.
  • Incubate tissue with secondary goat anti-rabbit AF488 antibody (Invitrogen, A11008, 1911238) diluted 1:200 in PBS 1% BSA solution for 1h at RT.
  • Wash cells 2x for 5min with PBS.
  • Wash tissue 2x for 3min with PBS 0.1% triton.
  • Wash tissue 2x for 3min with PBS.
  • DAPI counterstain for 5min at RT.
  • Mount slides with coverslips in Calbicochem (Millipore, 345789, lot 2682585).
  • Image acquisition with NIS-ELEMENTS (Nikon).

Experimental Notes:

ABIN1386389 did not label recombinantly expressed PKP2-FLAG in transfected HL1 cells. A hypothesis could be that antibody recognizes the murine protein in mouse heart samples but not the human sequence of the vector (transfected HL1).

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-Plakophilin 2 antibody (ABIN1386389)

Akdis, Medeiros-Domingo, Gaertner-Rommel, Kast, Enseleit, Bode, Klingel, Kandolf, Renois, Andreoletti, Akdis, Milting, Lüscher, Brunckhorst, Saguner, Duru: "Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls." in: Heart rhythm, Vol. 13, Issue 3, pp. 731-41, (2016) (PubMed).

Target Details for Plakophilin 2

Target: Plakophilin 2 (PKP2)

Alternative Name: Plakophilin 2 (PKP2 Products)

Synonyms: ARVD9 antibody, 1200008D14Rik antibody, 1200012P04Rik antibody, AA516617 antibody, Pkp2l antibody, plakophilin 2 antibody, PKP2 antibody, Pkp2 antibody

Background:

Synonyms: ARVD 9, ARVD-9, ARVD9, PKP 2, PKP2, PKP-2, Plakophilin-2. Plakophilin2, PKP2_HUMAN.

Background: Plakophilins 1, 2, 3 and 4 (PKP1-4) influence development and participate in linking cadherins to cytoskeletal intermediate filaments. Plakophilins 1-4 contain arm-repeat (armadillo) domains, and localize to nuclei and cell desmosomes (cell-cell junctions found in suprabasal layers of stratifying epithelia that undergo mechanical stress). Plakophilin-1 mediates increases in desmosomal protein content, desmosome assembly, and regulation of cell migration. Plakophilin-2 is important for desmosome assembly and is an essential morphogenic factor and architectural component of the heart. Plakophilin-3 plays a role in both desmosome-dependent adhesion and signaling pathways. Plakophilin-4 is a component of desmosomal adhesion plaques that regulates junctional plaque organization and cadherin function.

Gene ID: 5318

UniProt: Q99959

Pathways: Cell-Cell Junction Organization, SARS-CoV-2 Protein Interactome, The Global Phosphorylation Landscape of SARS-CoV-2 Infection