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MR1 antibody (AA 201-300)

MR1 Reactivity: Human ELISA, WB, IF Host: Mouse Monoclonal 5B5 unconjugated
Catalog No. ABIN516526
  • Target See all MR1 Antibodies
    MR1 (Major Histocompatibility Complex, Class I-Related (MR1))
    Binding Specificity
    • 11
    • 8
    • 6
    • 4
    • 2
    • 1
    • 1
    • 1
    • 1
    AA 201-300
    Reactivity
    • 34
    • 2
    • 1
    Human
    Host
    • 31
    • 3
    Mouse
    Clonality
    • 32
    • 2
    Monoclonal
    Conjugate
    • 14
    • 4
    • 3
    • 3
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    This MR1 antibody is un-conjugated
    Application
    • 23
    • 22
    • 4
    • 2
    • 2
    • 1
    • 1
    ELISA, Western Blotting (WB), Immunofluorescence (IF)
    Purpose
    Mouse monoclonal antibody raised against a partial recombinant MR1.
    Sequence
    TEPPLVRVNR KETFPGVTAL FCKAHGFYPP EIYMTWMKNG EEIVQEIDYG DILPSGDGTY QAWASIELDP QSSNLYSCHV EHCGVHMVLQ VPQESETIPL
    Cross-Reactivity
    Human
    Characteristics
    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.
    Clone
    5B5
    Isotype
    IgG1
  • Application Notes
    Optimal working dilution should be determined by the investigator.
    Restrictions
    For Research Use only
  • Validation #101752 (Immunocytochemistry)
    'Independent Validation' Badge
    by
    Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine
    No.
    #101752
    Date
    02/20/2018
    Antigen
    MR1
    Lot Number
    12045-5B5
    Method validated
    Immunocytochemistry
    Positive Control
    HEK293 cells transfected with human MR1 cDNA
    Negative Control
    HEK293 cells transfected with plasmid vector only
    Notes

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

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN516526
    Secondary Antibody
    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×.
    Experimental Notes

    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.

  • Validation #102826 (Immunoprecipitation)
    'Independent Validation' Badge
    by
    Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine
    No.
    #102826
    Date
    02/20/2018
    Antigen
    MR1
    Lot Number
    12045-5B5
    Method validated
    Immunoprecipitation
    Positive Control
    HEK293 cells transfected with human MR1 cDNA
    Negative Control
    HEK293 cells transfected with plasmid vector only
    Notes

    Passed. ABIN516526 immunoprecipitates human MR1 overexpressed by HEK293 cells.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN516526
    Secondary Antibody
    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).
    Experimental Notes

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

  • Validation #102827 (Flow Cytometry)
    'Independent Validation' Badge
    by
    Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine
    No.
    #102827
    Date
    02/20/2018
    Antigen
    MR1
    Lot Number
    12045-5B5
    Method validated
    Flow Cytometry
    Positive Control
    HEK293 cells transfected with human MR1 cDNA
    Negative Control
    HEK293 cells transfected with plasmid vector only
    Notes

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

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN516526
    Secondary Antibody
    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.
    Experimental Notes

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

  • Buffer
    In 1x PBS, pH 7.4
    Handling Advice
    Aliquot to avoid repeated freezing and thawing.
    Storage
    -20 °C
    Storage Comment
    Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing.
  • Target
    MR1 (Major Histocompatibility Complex, Class I-Related (MR1))
    Alternative Name
    MR1 (MR1 Products)
    Synonyms
    YFV antibody, hlals antibody, MGC154362 antibody, MR1 antibody, DKFZp468C1823 antibody, HLALS antibody, H2ls antibody, Hlals antibody, MHC class I antigen YF5 antibody, major histocompatibility complex, class I-related antibody, major histocompatibility complex, class I-related L homeolog antibody, YF5 antibody, MR1 antibody, mr1.L antibody, mr1 antibody, Mr1 antibody
    Background
    Full Gene Name: major histocompatibility complex, class I-related
    Synonyms: HLALS
    Gene 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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