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MR1 Antikörper (AA 201-300)

MR1 Reaktivität: Human ELISA, WB, IF Wirt: Maus Monoclonal 5B5 unconjugated
Produktnummer ABIN516526
  • Target Alle MR1 Antikörper anzeigen
    MR1 (Major Histocompatibility Complex, Class I-Related (MR1))
    Bindungsspezifität
    • 11
    • 8
    • 6
    • 4
    • 2
    • 1
    • 1
    • 1
    • 1
    AA 201-300
    Reaktivität
    • 37
    • 5
    • 4
    Human
    Wirt
    • 31
    • 6
    Maus
    Klonalität
    • 32
    • 5
    Monoklonal
    Konjugat
    • 15
    • 4
    • 3
    • 3
    • 3
    • 3
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Dieser MR1 Antikörper ist unkonjugiert
    Applikation
    • 23
    • 22
    • 5
    • 5
    • 2
    • 1
    • 1
    • 1
    ELISA, Western Blotting (WB), Immunofluorescence (IF)
    Verwendungszweck
    Mouse monoclonal antibody raised against a partial recombinant MR1.
    Sequenz
    TEPPLVRVNR KETFPGVTAL FCKAHGFYPP EIYMTWMKNG EEIVQEIDYG DILPSGDGTY QAWASIELDP QSSNLYSCHV EHCGVHMVLQ VPQESETIPL
    Kreuzreaktivität
    Human
    Produktmerkmale
    Antibody Reactive Against Recombinant Protein.
    Immunogen
    MR1 (NP_001522, 201 a.a. ~ 300 a.a) partial recombinant protein with GST tag. MW of the GST tag alone is 26 KDa.
    Klon
    5B5
    Isotyp
    IgG1
  • Applikationshinweise
    Optimal working dilution should be determined by the investigator.
    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Validierung #101752 (Immunocytochemistry)
    'Independent Validation' Siegel
    by
    Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine
    No.
    #101752
    Datum
    20.02.2018
    Antigen
    MR1
    Chargennummer
    12045-5B5
    Validierte Anwendung
    Immunocytochemistry
    Positivkontrolle
    HEK293 cells transfected with human MR1 cDNA
    Negativkontrolle
    HEK293 cells transfected with plasmid vector only
    Bewertung

    Passed. The MR1 antibody ABIN516526 specifically labels the targeted antigen in HEK293 ectopically expressing human MR1 in ICC.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN516526
    Sekundärantikörper
    FITC-conjugated donkey anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 715-096-151, lot 76750)
    Full Protocol
    • Grow HEK293 cells in DMEM medium (Lonza, 12-614F, lot 0000618582) supplemented with serum (Hyclone, SH30071.03, lot AAG205460) and antibiotics (Hyclone, SV30010, lot J150013), at 37°C and 5% CO2 dish to 70-90% confluency.
    • Transfect cells with pCDNA 3.1 neo (-) (Invitrogen) containing human MR1 cDNA (Genecopoeia) using Polyethylenimine (Polysciences, 23966) following the manufacturer´s instructions.
    • Plate human MR1-expressing HEK293 cells in sterile glass-bottom 35mm dishes coated with collagen (MatTek, P35GCol-1.5-14-C) to 50-80% confluency.
    • Wash cells PBS.
    • Fix cells with 4% Paraformaldehyde for 15min at RT.
    • Block cells with blocking buffer (1x PBS, 5% donkey serum, 0.3% Triton X-100) for 1h at RT.
    • Incubate cells with primary mouse anti-MR1 antibody (antibodies-online, ABIN516526, lot 12045-5B5) diluted 1:50 in dilution buffer (1x PBS, 1% BSA, 0.3% Triton X-100) and incubated ON at 4°C.
    • Wash cells 3x with PBS.
    • Incubated with secondary FITC-conjugated donkey anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 715-096-151, lot 76750) diluted 1:50 in dilution buffer for 1h at RT.
    • Wash cells 3x with PBS.
    • To stain the nucleus, immerse cells in PBS containing Hoechst (1:2000) for 5min.
    • Just prior to confocal analysis, place cells in mounting medium (10mM Tris pH8.5, 2% DABCO).
    • Image cells on an Olympus 2 confocal/two-photon microscope imaging system using an oil immersion lens at 60×.
    Anmerkungen

    Staining with ABIN516526 shows a perinuclear pattern, suggesting MR1 localizes in the endoplasmic reticulum. No signal was detected in sample negative control tissue and the secondary antibody only control.

  • Validierung #102826 (Immunoprecipitation)
    'Independent Validation' Siegel
    by
    Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine
    No.
    #102826
    Datum
    20.02.2018
    Antigen
    MR1
    Chargennummer
    12045-5B5
    Validierte Anwendung
    Immunoprecipitation
    Positivkontrolle
    HEK293 cells transfected with human MR1 cDNA
    Negativkontrolle
    HEK293 cells transfected with plasmid vector only
    Bewertung

    Passed. ABIN516526 immunoprecipitates human MR1 overexpressed by HEK293 cells.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN516526
    Sekundärantikörper
    goat anti-rabbit Dye-IR800 conjugated antibody (Advansta, R-05060-250, lot 17083179)
    Full Protocol
    • Grow HEK293 cells in DMEM medium (Lonza, 12-614F, lot 0000618582) supplemented with serum (Hyclone, SH30071.03, lot AAG205460) and antibiotics (Hyclone, SV30010, lot J150013), at 37°C and 5% CO2 dish to 70-90% confluency.
    • Transfect cells with pCDNA 3.1 neo (-) (Invitrogen) containing human MR1 cDNA (Genecopoeia) using Polyethylenimine (Polysciences, 23966) following the manufacturer´s instructions.
    • Lyse cells in cold lysis buffer (10mM Tris pH7.4, 150mM NaCl, 0.5mM EDTA, 2% CHAPS).
    • Determine total protein content of the lysates using Commassie Protein Assay Reagent (Thermo Scientific, 1856209, lot NL179252).
    • Immobilize 100µl of protein G-conjugated Sepharose beads (Pierce, product 20399, lot RI239318) ON at 4°C with
      • 2.5µg mouse anti-MR1 antibody (antibodies-online, ABIN2665876, lot B177559),
      • 2.5µg mouse anti-MR1 antibody (antibodies-online, ABIN516526, lot12045-5B5),
      • 2.5µg rabbit anti-MR1 antibody (antibodies-online, ABIN1537116, lot SA111213CH),
      • 2.5µg mouse IgG2a antibody (Biolegend, 400202, lot B153642),
      • 2.5µg mouse IgG1 antibody (BD, 555746, lot 3221830), or
      • 2.5µg rabbit IgG antibody (Santa Cruz Biotechnology, SC-5560, lot E0609).
    • Incubate 500µg of the cell lysates with 2.5µg of antibody-bead conjugate ON at 4°C.
    • Wash lysates 4x with PBS.
    • Denature beads for 5min at 95°C in 60µl Laemmli SDS sample buffer and subsequently separate them on a SDS-PAGE gel using Acrylamide/Bis Premixed (Bio-Rad, 61-0125, lot 260000477) for 2-3h at 100V.
    • Transfer proteins onto PVDF membrane (Millipore, IPVH00010, lot K5AA6843U) with a Western blotting system for ON at 4°C at 150mA.
    • Block the membrane with blocking buffer (2% BSA/PBS/0.05%Tween-20) for 1h at RT.
    • Incubate membrane with primary rabbit anti-MR1 antibody (antibodies-online ABIN1537116, lot SA111213CH) diluted 1:1000 in blocking buffer ON at 4°C.
    • Wash membrane 3x for 10min with PBS/0.05%Tween-20.
    • Incubate membrane with secondary goat anti-rabbit Dye-IR800 conjugated antibody (Advansta, R-05060-250, lot 17083179) diluted 1:10000 in PBS/0.05% Tween-20 for 1h at RT.
    • Wash membrane 3x for 10 min with PBS/0.05% Tween-20.
    • Reveal protein bands using an Odyssey imaging system (LI-COR Biosciences).
    Anmerkungen

    The human MR1 antibody ABIN516526, but not the isotype control, immunoprecipitates with human MR1 overexpressed by HEK293 cells.

  • Validierung #102827 (Flow Cytometry)
    'Independent Validation' Siegel
    by
    Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine
    No.
    #102827
    Datum
    20.02.2018
    Antigen
    MR1
    Chargennummer
    12045-5B5
    Validierte Anwendung
    Flow Cytometry
    Positivkontrolle
    HEK293 cells transfected with human MR1 cDNA
    Negativkontrolle
    HEK293 cells transfected with plasmid vector only
    Bewertung

    Passed. ABIN2665876 recognizes human MR1 overexpressed by HEK293 cells and can be used in flow cytometry.

    'Independent Validation' Siegel
    Validierungsbilder
    Protokoll
    Primärantikörper
    ABIN516526
    Sekundärantikörper
    PE-conjugated rabbit anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 115-116-146, lot 120701)
    Full Protocol
    • Grow HEK293 cells in DMEM medium (Lonza, 12-614F, lot 0000618582) supplemented with serum (Hyclone, SH30071.03, lot AAG205460) and antibiotics (Hyclone, SV30010, lot J150013), at 37°C and 5% CO2 dish to 70-90% confluency.
    • Transfect cells with pCDNA 3.1 neo (-) (Invitrogen) containing human MR1 cDNA (Genecopoeia) using Polyethylenimine (Polysciences, 23966) following the manufacturer´s instructions.
    • Surface staining:
      • Wash 0.5x106 cells 3x with HBSS/0.1% BSA.
      • Incubate cells with mouse anti-MR1 antibody (antibodies-online, ABIN516526, lot 12045-5B5) diluted 1:100 or mouse anti-MR1 antibody (antibodies-online, ABIN2665876, lot B177559) diluted 1:100 for 30min on ice.
      • Wash cells 3x with HBSS/0.1% BSA.
      • Incubate cells with a PE-conjugated rabbit anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 115-116-146, lot 120701) diluted 1:100 for 30min on ice.
    • Total cell staining:
      • Fix 0.5x106 cells in 1% paraformaldehyde for 10min at RT.
      • Wash cells with HBSS/BSA.
      • Permeabilize cells in HBSS/BSA with 0.1% saponin for 10min at RT.
      • Incubate cells with mouse anti-MR1 antibody (antibodies-online, ABIN516526, lot 12045-5B5) diluted 1:100 or mouse anti-MR1 antibody (antibodies-online, ABIN2665876, lot B177559) dilute 1:100 for 30min at RT in the presence of 0.1% saponin.
      • Wash cells 3x with HBSS/0.1% BSA/0.1% saponin.
      • Incubate cells with PE-conjugated rabbit anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 115-116-146, lot 120701) diluted 1:100 for 30min at RT.
    • Acquire flow cytometry data using a flow cytometry using LSR 4 (BD Biosciences) followed data analysis using FlowJo software.
    Anmerkungen

    The human MR1 antibody ABIN2665876 shows specific staining for human MR1.

  • Buffer
    In 1x PBS, pH 7.4
    Handhabung
    Aliquot to avoid repeated freezing and thawing.
    Lagerung
    -20 °C
    Informationen zur Lagerung
    Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing.
  • Target
    MR1 (Major Histocompatibility Complex, Class I-Related (MR1))
    Andere Bezeichnung
    MR1 (MR1 Produkte)
    Synonyme
    YFV antikoerper, hlals antikoerper, MGC154362 antikoerper, MR1 antikoerper, DKFZp468C1823 antikoerper, HLALS antikoerper, H2ls antikoerper, Hlals antikoerper, MHC class I antigen YF5 antikoerper, major histocompatibility complex, class I-related antikoerper, major histocompatibility complex, class I-related L homeolog antikoerper, YF5 antikoerper, MR1 antikoerper, mr1.L antikoerper, mr1 antikoerper, Mr1 antikoerper
    Hintergrund
    Full Gene Name: major histocompatibility complex, class I-related
    Synonyms: HLALS
    Gen-ID
    3140
    NCBI Accession
    NM_001531
    Pathways
    Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Cancer Immune Checkpoints
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