MR1 anticorps (Major Histocompatibility Complex, Class I-Related) (AA 201-300)

Details for Product anti-MR1 Antibody No. ABIN516526
Antigène
  • YFV
  • hlals
  • MGC154362
  • MR1
  • DKFZp468C1823
  • HLALS
  • H2ls
  • Hlals
  • MHC class I antigen YF5
  • major histocompatibility complex, class I-related
  • major histocompatibility complex, class I-related L homeolog
  • YF5
  • MR1
  • mr1.L
  • mr1
  • Mr1
Épitope
AA 201-300
12
9
5
4
3
3
1
1
1
1
1
Reactivité
Humain
44
3
1
1
Hôte
Souris
40
5
Clonalité (Clone)
Monoclonal ()
Conjugué
Cet anticorp MR1 est non-conjugé
4
3
3
2
2
2
1
1
1
1
1
1
Application
ELISA, Flow Cytometry (FACS), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunoprecipitation (IP), Western Blotting (WB)
34
25
4
2
2
1
1
Options
'Independent Validation' signe
Antigène MR1
Numéro du lot 12045-5B5
Application validée Immunocytochemistry
Contrôle positif HEK293 cells transfected with human MR1 cDNA
Contrôle négative HEK293 cells transfected with plasmid vector only
Conclusion

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

Anticorps primaire ABIN516526
Anticorps secondaire FITC-conjugated donkey anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 715-096-151, lot 76750)
Protocole
  • 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×.
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 Images
Immunocytochemistry validation image for anti-Major Histocompatibility Complex, Class I-Related (MR1) (AA 201-300) antibody (ABIN516526) Human MR1-expressing HEK293 cells were stained with MR1 antibody ABIN516526 and a FIT...
'Independent Validation' signe
Antigène MR1
Numéro du lot 12045-5B5
Application validée Immunoprecipitation
Contrôle positif HEK293 cells transfected with human MR1 cDNA
Contrôle négative HEK293 cells transfected with plasmid vector only
Conclusion

Passed. ABIN516526 immunoprecipitates human MR1 overexpressed by HEK293 cells.

Anticorps primaire ABIN516526
Anticorps secondaire goat anti-rabbit Dye-IR800 conjugated antibody (Advansta, R-05060-250, lot 17083179)
Protocole
  • 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).
Notes

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

Validation Images
Immunoprecipitation validation image for anti-Major Histocompatibility Complex, Class I-Related (MR1) (AA 201-300) antibody (ABIN516526) Lysates from human MR1-expressing HEK293 cells were immunoprecipitated by antibodies ...
'Independent Validation' signe
Antigène MR1
Numéro du lot 12045-5B5
Application validée Flow Cytometry
Contrôle positif HEK293 cells transfected with human MR1 cDNA
Contrôle négative HEK293 cells transfected with plasmid vector only
Conclusion

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

Anticorps primaire ABIN516526
Anticorps secondaire PE-conjugated rabbit anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 115-116-146, lot 120701)
Protocole
  • 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.
Notes

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

Validation Images
Flow Cytometry validation image for anti-Major Histocompatibility Complex, Class I-Related (MR1) (AA 201-300) antibody (ABIN516526) Human MR1-expressing HEK293 cells (MR1) and vector control cells (Vc) were stained wi...
Immunogène MR1 (NP_001522, 201 a.a. ~ 300 a.a) partial recombinant protein with GST tag. MW of the GST tag alone is 26 KDa.
Immunogen sequence: TEPPLVRVNR KETFPGVTAL FCKAHGFYPP EIYMTWMKNG EEIVQEIDYG DILPSGDGTY QAWASIELDP QSSNLYSCHV EHCGVHMVLQ VPQESETIPL
Clone 5B5
Isotype IgG1 kappa
 Réactivité croisée Humain
Plasmids, Primers & others Plasmids, Primers & others MR1 products on genomics-online (e.g. as negative or positive controls)
Antigène
Autre désignation MR1 (MR1 Antibody Extrait)
Sujet Full Gene Name: major histocompatibility complex, class I-related
Synonyms: HLALS
ID gène 3140
NCBI Accession NP_001522, NM_001531
Pathways Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Cancer Immune Checkpoints
Indications d'application Optimal working dilution should be determined by the investigator.
Commentaires

Product Quality tested by: Antibody Reactive Against Recombinant Protein.

Restrictions For Research Use only
Buffer In 1x PBS, pH 7.4
Conseil sur la manipulation Aliquot to avoid repeated freezing and thawing.
Stock -20 °C
Stockage commentaire Store at -20 °C or lower. Aliquot to avoid repeated freezing and thawing.
Images (Fournisseur)
 image for anti-Major Histocompatibility Complex, Class I-Related (MR1) (AA 201-300) antibody (ABIN516526) Western Blot detection against Immunogen (36.74 KDa) .
Immunofluorescence (IF) image for anti-Major Histocompatibility Complex, Class I-Related (MR1) (AA 201-300) antibody (ABIN516526) Immunofluorescence of monoclonal antibody to MR1 on HeLa cell. [antibody concentratio...
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