Plakophilin 2 Antikörper (N-Term)

Produktnummer ABIN2786784

Produktdetails anti-Plakophilin 2 Antikörper

Target: Plakophilin 2 (PKP2)

Bindungsspezifität: N-Term

Reaktivität: Human, Ratte, Maus, Rind (Kuh), Hund, Pferd

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser Plakophilin 2 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunohistochemistry (IHC)

Sequenz: HPLRRLEISP DSSPERAHYT HSDYQYSQRS QAGHTLHHQE SRRAALLVPP

Homologie: Cow: 77%, Dog: 79%, Horse: 86%, Human: 100%, Mouse: 86%, Rat: 77%

Produktmerkmale: This is a rabbit polyclonal antibody against PKP2. It was validated on Western Blot using a cell lysate as a positive control.

Aufreinigung: Affinity Purified

Immunogen: The immunogen is a synthetic peptide directed towards the N terminal region of human PKP2

Anwendungsinformationen

Applikationshinweise: Optimal working dilutions should be determined experimentally by the investigator.

Kommentare:

Antigen size: 837 AA

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #103654 (Western Blotting)

by: Gencardio (IDIBGI)

No.: #103654

Datum: 13.03.2019

Antigen: PKP2

Chargennummer: QC27639-43357

Validierte Anwendung: Western Blotting

Positivkontrolle:

HL-1 cell extracts (from AT-1 mouse atrial cardiomyocyte tumor lineage)

Extracts from HEK293 cells transfected with human PKP2-FLAG expressing plasmid

Extract of mouse heart tissue (endogenous PKP2 expected molecular weight 88KDa)

Extract of human heart tissue (endogenous PKP2 expected molecular weight 91KDa)

Negativkontrolle:

Extracts from untransfected HEK293 cells

Bewertung:

ABIN2786784 specifically recognizes PKP2 in cell extracts from HEK-293 overexpressing human PKP2 protein and endogenous PKP2 in human heart tissue but not endogenous mouse PKP2 neither in HL-1 cells nor mouse tissue.

Validierungsbilder

Western blot of lysates from HEK293 cells transfected with FLAG-tagged PKP2 (1), non-transfected HEK293 cells (2), non-transfected HL1 cells (3) HEK293 cells transfected with ABIN2786784 or an anti-FLAG tag antibody. In addition, lysates from human (4) and mouse heart tissue (5, 6) were incubated with ABIN2786784.

Protokoll

Primärantikörper: ABIN2786784

Sekundärantikörper: mouse anti-FLAG-tag antibody (Sigma, monoclonal anti-flag, F316, lot SLBT7654)

Full Protocol:

• Grow HL-1 cells in Claycomb medium (Sigma, 51800C, lot SLBX8700) supplemented with 10% FBS (Gibco, 10270-106, lot 42G5178K), 1% Penicillin-Streptomycin (Sigma, P4333, lot 048M4874V) and 1% Glutamax (Gibco, 35050-061, 2037045), at 37°C and 5% CO₂ in 2ml on a 6 well plate to 95% confluency.
• Grow HEK293 cells in DMEM/High Modified medium (GE Life Science, SH30285.01, lot AC14565263) supplemented with 10% FBS, 1% Penicillin-Streptomycin and 1% Glutamax (Gibco, 35050-061, 2037045), at 37°C and 5% CO₂ in 2ml on a 6 well plate to 95% confluency.
• Transfect HEK293 cells with PKP2a plasmid (p915 PKP2a-pFLAG-cmv5a; provided by Dr. Green KJ) using Lipofectamin 2000 (Invitrogen, 11668-019, lot 2022713) following the manufacturer´s instructions.
• Lyse approximately 1.2x10⁶ cells per well in 200µl 1% SDS (Sigma, 75746, lot STBG4187V).
• Homogenate whole heart of transgenic and wt heart mouse tissue with M-Per reagent (Thermo, 78501, lot NC168900) and mechanical disgregation using a Dounce homogenizer.
• Incubate for the lysates from 15min at 95°C.
• Vortex for 15min.
• Determine total protein content of the lysates using Pierce BCA Protein Assay Kit (Thermo Scientific, 23227, lot TC263613).
• Denature 15µg (HEK293 cell lysates), 106µg (HL-1 cell lysates), and 100µg (mouse heart tissue lysates) of total protein for 5min at 95°C in 45µl using 6x Laemmli SDS sample buffer and subsequently separate them on a 8% acrylamide gel in an electrophoresis chamber for 30min at 80V and 1h at 120V.
• Transfer proteins onto Amersham Hybond P 0.45 PVDF blotting membrane (GE Life Science, 10600023, lot A16954274) for 2h at 80V.
• Block the membrane with 5% NFM in PBST for 1h at RT.
• Incubation with primary
• • rabbit anti-Plakophilin 2 (PKP2) (N-Term) antibody (antibodies-online, ABIN2786784, lot QC27639-43357) diluted 1:800 in 5% NFM in PBS ON at 4°C or
  • mouse anti-FLAG-tag antibody (Sigma, monoclonal anti-flag, F316, lot SLBT7654) diluted 1:x in 5% NFM in PBS ON at 4°C.
• Wash membrane 2x for 5’ with PBST and 2x for 5min with PBS.
• Block the membrane with 5% NFM in PBST for 1h at RT.
• Incubation with secondary anti-rabbit (Jackson ImmunoResearch, 111-035-003, lot # 135002) diluted 1:10000 in 5% NFM in PBS for 1h at RT.
• Wash membrane 2x for 5min with PBST and 2x for 5min with PBS.
• Reveal protein bands using Perce ECL Western (Thermo Scientific, 32106, TE265714) with Amersham Hyperfilm ECL (GE Healthcare Limited, 28906835, 65901).

Anmerkungen:

HEK293 cells with no endogenous PKP2 expression have been transfected with human FLAG-tagged PKP2 as a positive control. HL-1 cells express endogenous mouse PKP2.

Handhabung

Format: Liquid

Konzentration: Lot specific

Buffer: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handhabung: Avoid repeated freeze-thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: For short term use, store at 2-8°C up to 1 week. For long term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles.

Details zu Plakophilin 2

Target: Plakophilin 2 (PKP2)

Andere Bezeichnung: PKP2 (PKP2 Produkte)

Synonyme: ARVD9 antikoerper, 1200008D14Rik antikoerper, 1200012P04Rik antikoerper, AA516617 antikoerper, Pkp2l antikoerper, plakophilin 2 antikoerper, PKP2 antikoerper, Pkp2 antikoerper

Hintergrund: This gene encodes a member of the arm-repeat (armadillo) and plakophilin gene families. Plakophilin proteins contain numerous armadillo repeats, localize to cell desmosomes and nuclei, and participate in linking cadherins to intermediate filaments in the
Alias Symbols: ARVD9
Protein Interaction Partner: NDEL1, MTUS2, CEP250, DROSHA, BMI1, EED, RNF2, YWHAB, CLK2, YWHAE, WHSC1, UBC, SMAD9, YWHAQ, CUL3, MARK3, POLR3A, YWHAG, PKP2, JUP, KRT18, KRT5, DSG2, DSG1, DSP, DSC2, DSC1, CTNNB1, GTF2B, SFN,
Protein Size: 837

Molekulargewicht: 93 kDa

Gen-ID: 5318

NCBI Accession: NM_001005242, NP_001005242

UniProt: A0AV37

Pathways: Cell-Cell Junction Organization, SARS-CoV-2 Protein Interaktom, Phosphorylierungen bei SARS-CoV-2 Infektion