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NPM1 Antikörper (C-Term)

NPM1 Reaktivität: Human WB, IF, IHC (p), IP, FACS Wirt: Kaninchen Polyclonal unconjugated
Produktnummer ABIN1737584
  • Target Alle NPM1 Antikörper anzeigen
    NPM1 (Nucleophosmin (Nucleolar phosphoprotein B23, Numatrin) (NPM1))
    Bindungsspezifität
    • 21
    • 15
    • 15
    • 12
    • 10
    • 9
    • 7
    • 7
    • 6
    • 5
    • 4
    • 3
    • 3
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    AA 250-298, C-Term
    Reaktivität
    • 147
    • 105
    • 76
    • 13
    • 12
    • 10
    • 9
    • 9
    • 7
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    Human
    Wirt
    • 141
    • 29
    • 1
    • 1
    Kaninchen
    Klonalität
    • 138
    • 35
    Polyklonal
    Konjugat
    • 99
    • 15
    • 6
    • 6
    • 6
    • 6
    • 6
    • 6
    • 5
    • 3
    • 3
    • 3
    • 3
    • 3
    • 3
    Dieser NPM1 Antikörper ist unkonjugiert
    Applikation
    • 100
    • 61
    • 43
    • 40
    • 35
    • 28
    • 27
    • 15
    • 13
    • 12
    • 7
    • 7
    • 7
    • 5
    • 5
    • 4
    • 3
    • 3
    • 1
    Western Blotting (WB), Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP), Flow Cytometry (FACS)
    Spezifität
    Reacts with the mutant form of Nucleophosmin
    Aufreinigung
    Antigen affinity purified
    Immunogen
    Synthetic peptide made to a C-terminal region of human mutant nucleophosmin (within residues 250-294) [AAW67752]
  • Applikationshinweise

    Working dilution: Optimal dilution should be determined by the end user.
    The following are guidelines only :
    FC(1:100) ICC/IF(1:50 - 1:100) IHC - P(1:50 - 1:100) IP(1:50) WB(1:1000)

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Validierung #101096 (Western Blotting)
    'Independent Validation' Siegel
    by
    Institute of Hematology and Blood Transfusion, Prague
    No.
    #101096
    Datum
    17.05.2017
    Antigen
    NPM1
    Chargennummer
    Validierte Anwendung
    Western Blotting
    Positivkontrolle
    OCI-AML3 cells containing the NPM1 mutation type A, HEK-293T transfected with GFP-NPM mutA
    Negativkontrolle
    HL-60 cells
    Bewertung
    Passed. ABIN1737584 specifically recognizes mutated nucleophosmin in OCI-AML3 cells and NPMmut transfected HEK-293T cells.
    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN1737584
    Sekundärantikörper
    goat-anti rabbit antibody (LifeTechnologies, cat. No.31460)
    Full Protocol
    • Grow OCI-AML3 (DSMZ) cells in MEM medium (Sigma-Aldrich, M8042, lot RNBF-0384) supplemented with 20% FBS (Biochrom, S0615, lot 0513C) and penicillin-streptomycin (Sigma-Aldrich, P4333, lot 066M4791V) at 37°C and 5% CO2 in 5ml on a tissue culture dish to 1.5x106 cells/ml.
    • Grow HL-60 (ECACC) cells RPMI medium (Sigma-Aldrich, R0883, lot RNBF-7081) supplemented with 10% FBS and penicillin-streptomycin at 37°C and 5% CO2 in 5ml on a tissue culture dish to 1.5x106 cells/ml.
    • Grow HEK-293T cells on DMEM medium (Sigma-Aldrich, D8062, lot RNBF-4838) supplemented with 10% FBS and penicillin-streptomycin at 37°C and 5% CO2 in 5ml on a tissue culture dish to 60% confluency.
    • Transfect HEK-293T cells with plasmid containing gene for GFP-NPMwt or GFP-NPMmutA using jetPRIME transfection reagent (Polyplus Transfection, 114-15, lot 1240106I17) following the manufacturer´s instructions.
    • Collect 3-5x106 cells directly in 100μl 1x Laemmli SDS sample buffer, and boil them for 5min at 95°C.
    • Centrifuge samples at 200.000g/4°C for 4h. Take of the supernatant and store at -20°C.
    • Separate 10µl of each sample on a freshly cast Tris HCl pH8.8 12,5% SDS-PAGE in a Mini-PROTEAN Tetra Cell System for 1h at 200V/4°C.
    • Transfer proteins onto PVDF membrane (GE Healthcare, 10600023, lot A10125999) with a semi-dry blotting system for 1.5h at 100mA.
    • Block the membrane with 5% non-fat dry milk in PBS-Tween (0.5% v/v) for 1h at RT.
    • Incubation the membrane with primary
      • rabbit anti-mutant nucleophosmin (antibodies-online, ABIN1737584) diluted 1:1000 in 5% non-fat dry milk in PBSTON at 4°C,
      • mouse anti-NPM (Santa Cruz, sc-70392) recognizing the N-terminus of NPM protein (i.e. both the NPM wild-type and NPM mutant type A) diluted 1:500 in 5% non-fat dry milk in PBSTON at 4°C, or
      • mouse anti-NPM (Santa Cruz, sc-271737) recognizing the C-terminus of NPM wild-type protein diluted 1:500 in 5% non-fat dry milk in PBSTON at 4°C.
    • Wash the membrane 6x for 5min in PBST.
    • Incubate the membrane with secondary HRP-conjugated
      • goat-anti rabbit antibody (LifeTechnologies, cat. No.31460) diluted 1:50.000 in PBST for 1h at RT or
      • goat-anti mouse antibody (LifeTechnologies, cat. No.31430) diluted 1:50.000 in PBST for 1h at RT.
    • Wash membrane 6x for 5 min with PBS-Tween.
    • Visualize protein bands using ECL Plus detection reagent (GE Healthcare, RPN2232, lot 9732255) on a G-box iChemi XT4 digital imaging device (Syngene Europe).
    Anmerkungen
    The nucleophosmin antibody ABIN1737584 reveals a protein of the expected molecular weight in lysates of OCI-AML3 and of HEK-293T transfected with GFP-NPM mutA. The protein band is but not visible in the HL-60 negative control. A weak band from OCI-AML3 detected by an anti-NPMwt antibody confirms the concurrent presence of both NPMmutA and NPMwt in the OCI-AML3 cell line.
  • Validierung #101288 (Immunofluorescence)
    'Independent Validation' Siegel
    by
    Institute of Hematology and Blood Transfusion, Prague
    No.
    #101288
    Datum
    17.05.2017
    Antigen
    NPM1
    Chargennummer
    Validierte Anwendung
    Immunofluorescence
    Positivkontrolle
    OCI-AML3 cells containing the NPM1 mutation type A
    Negativkontrolle
    HL-60 cells
    Bewertung
    Passed. ABIN1737584 specifically recognizes mutated nucleophosmin in sample.
    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN1737584
    Sekundärantikörper
    AlexaFluor488-conjugated goat anti-rabbit antibody (LifeTechnologies, A31627, lot 1440410)
    Full Protocol
    • Grow OCI-AML3 (DSMZ) cells in MEM medium (Sigma-Aldrich, M8042, lot RNBF-0384) supplemented with 20% FBS (Biochrom, lot 0513C) and penicillin-streptomycin (Sigma-Aldrich, P4333, lot 066M4791V) at 37°C and 5% CO2 in 5ml on a tissue culture dish to 1.5x106 cells/ml.
    • Grow HL-60 (ECACC) cells RPMI medium (Sigma-Aldrich, R0883, lot RNBF-7081) supplemented with 10% FBS and penicillin-streptomycin at 37°C and 5% CO2 in 5ml on a tissue culture dish to 1.5x106 cells/ml.
    • Wash 106 cells with PBS, resuspend them in 150µl PBS and apply the suspension on a coverslip in a humidified chamber for 20min.
    • Aspirate PBS with thick filter paper.
    • Apply 4% paraformaldehyde for 1h at RT.
    • Wash the coverslip briefly with PBS and apply 0.2% Triton X-100 for 10min at RT.
    • Wash the coverslip 3x for 5 min with PBS-Tween (PBS, Tween 20 0.2% v/v).
    • Incubate the coverslip with image iT-signal enhancer (LifeTechnologies, from A31627 kit) for 30min at RT.
    • Wash the coverslip 3x for 5min with PBST.
    • Incubate the coverslip with primary rabbit anti-mutant nucleophosmin(antibodies-online, ABIN1737584) diluted 1:100 and mouse monoclonal anti-NPM (Santa Cruz, sc-70392) diluted 1:50 in PBST for 1h at RT.
    • Wash the coverslip 3x for 5 min with PBST.
    • Incubation the coverslip with secondary AlexaFluor488-conjugated goat anti-rabbit antibody (LifeTechnologies, A31627, lot 1440410), AlexaFluor647-conjugated goat anti-mouse antibody (LifeTechnologies, A31625, lot 1626792) diluted 1:200 and 1µM Hoechst33342 in PBST for 1h at RT in the dark.
    • Wash the membrane 2x for 5min with PBST.
    • Wash the membranc for 5 min in PBS.
    • Apply 10µl of ProLong Gold Antifade Mountant (LifeTechnologies, P36930) on a microscope slide and attach it to a coverslip. Dry ON.
    • Visualize the fluorescence signal under confocal laser scanning microscope FluoView FV1000 (Olympus Corporation) at 60x/1.35 oil objective.
    Anmerkungen
    The nucleophosmin antibody ABIN1737584 reveals a protein of the expected (cytoplasmic) localization of antigen in OCI-AML3 cell line. The protein is only visible in the positive but not the negative control (HL-60).
  • Validierung #101289 (Flow Cytometry)
    'Independent Validation' Siegel
    by
    Institute of Hematology and Blood Transfusion, Prague
    No.
    #101289
    Datum
    17.05.2017
    Antigen
    NPM1
    Chargennummer
    Validierte Anwendung
    Flow Cytometry
    Positivkontrolle
    OCI-AML3 cells containing the NPM1 mutation type A
    Negativkontrolle
    HL-60 cells
    Bewertung
    Passed. ABIN1737584 specifically recognizes mutated nucleophosmin in OCI-AML3 cells.
    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN1737584
    Sekundärantikörper
    AlexaFluor488-conjugated goat anti-rabbit antibody (LifeTechnologies, A31627, lot 1440410)
    Full Protocol
    • Grow OCI-AML3 (DSMZ) cells in MEM medium (Sigma-Aldrich, M8042, lot RNBF-0384) supplemented with 20% FBS (Biochrom, S0615, lot 0513C) and penicillin-streptomycin (Sigma-Aldrich, P4333, lot 066M4791V) at 37°C and 5% CO2 in 5ml on a tissue culture dish 1.5x106 cells/ml.
    • Grow HL-60 (ECACC) cells RPMI medium (Sigma-Aldrich, R0883, lot RNBF-7081) supplemented with 10% FBS and penicillin-streptomycin at 37°C and 5% CO2 in 5ml on a tissue culture dish 1.5x106 cells/ml.
    • Wash 5x105 cells with PBS, resuspend them in 200µl of 4% paraformaldehyde.
    • Incubate for 15min at RT.
    • Centrifuge 5min/500g/RT, aspirate the supernatant.
    • Resuspend cells in 50µl of ice-cold PBS and add 450µl of ice-cold methanol.
    • Store resuspended cells at -20°C for at least 30min (max 30 days).
    • Wash the cells 2x with PBS-BSA (1% BSA in PBS) and centrifuge them for 5min/500g.
    • Resuspend cells in 200µl PBS-BSA.
    • Incubate cells for 20min at RT.
    • Incubate cells with primary antibody
      • rabbit anti-mutant nucleophosmin(antibodies-online, ABIN1737584) diluted 1:100 in PBS-BSA for 1h at RT or
      • normal rabbit IgG (Santa Cruz, sc-2027) as negative staining control diluted 1:100 in PBS-BSA for 1h at RT.
    • Wash the cells 2x with PBS-BSA (1% BSA in PBS) and centrifuge them for 5min/500g.
    • Resuspend cells in 200µl PBS-BSA.
    • Incubate cells with secondary AlexaFluor488-conjugated goat anti-rabbit antibody (LifeTechnologies, A31627, lot 1440410) diluted 1:200 in PBS-BSA for 1h at RT.
    • Wash the cells 2x with PBS-BSA (1% BSA in PBS) and centrifuge them for 5min/500g.
    • Resuspend pellet in 300µl PBS and acquire AlexaFluor488 signal on flow-cytometer (BD Fortessa).
    Anmerkungen
    The nucleophosmin antibody ABIN1737584 reveals a strong fluorescence signal from OCI-AML3 cell line. Only moderate signal is obtained from the negative control (HL-60) and no signal is detected with non-specific primary antibody (normal rabbit IgG).
  • Format
    Liquid
    Konzentration
    1 mg/mL
    Buffer
    PBS pH 7.4, 0.02 % sodium azide
    Konservierungsmittel
    Sodium azide
    Vorsichtsmaßnahmen
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Lagerung
    4 °C/-20 °C
    Informationen zur Lagerung
    Short term storage at +4°C. For extended periods store in aliquots at -20°C. Antibodies are guaranteed for 6 month from date of receipt.
    Haltbarkeit
    6 months
  • Target
    NPM1 (Nucleophosmin (Nucleolar phosphoprotein B23, Numatrin) (NPM1))
    Andere Bezeichnung
    Nucleophosmin (NPM1 Produkte)
    Synonyme
    B23 antikoerper, NPM antikoerper, NO38 antikoerper, Npm antikoerper, B23NP antikoerper, npm4 antikoerper, npm antikoerper, CG7917 antikoerper, CRP1 antikoerper, Crp1 antikoerper, Dmel\\CG7917 antikoerper, dNLP antikoerper, dNLP-S antikoerper, p22 antikoerper, npm1 antikoerper, sb:cb487 antikoerper, nucleophosmin 1 antikoerper, nucleophosmin (nucleolar phosphoprotein B23, numatrin) L homeolog antikoerper, nucleophosmin 1b antikoerper, Nucleophosmin antikoerper, nucleophosmin antikoerper, nucleophosmin (nucleolar phosphoprotein B23, numatrin) antikoerper, Nucleoplasmin antikoerper, nucleophosmin 1a antikoerper, NPM1 antikoerper, Npm1 antikoerper, npm1.L antikoerper, npm1b antikoerper, npm antikoerper, Nlp antikoerper, npm1a antikoerper
    Hintergrund
    Nucleophosmin (NPM) is a multi-functional protein that is involved in many cellular activities including cell growth, proliferation and transformation. It is frequently overexpressed in solid tumors, and has recently been found to be mutated and aberrantly localized in the cytoplasm of leukaemic blasts in patients with acute myeloid leukemia (AML).
    Gen-ID
    4869
    Pathways
    Ribonucleoprotein Complex Subunit Organization, Ribosome Assembly
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