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

Details for Product anti-MR1 Antibody No. ABIN516526, Supplier: Log in to see
Antigen
  • 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
Alternatives
anti-Human MR1 antibody for ELISA
Epitope
AA 201-300
12
9
5
4
3
3
1
1
1
1
1
Reactivity
Human
44
9
7
1
Host
Mouse
34
11
Clonality (Clone)
Monoclonal ()
Conjugate
This MR1 antibody is un-conjugated
4
4
3
3
3
2
Application
ELISA, Flow Cytometry (FACS), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunoprecipitation (IP), Western Blotting (WB)
34
25
8
5
3
2
2
1
1
Options
Supplier
Log in to see
Supplier Product No.
Log in to see
'Independent Validation' Badge
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.

Primary Antibody ABIN516526
Secondary Antibody FITC-conjugated donkey anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 715-096-151, lot 76750)
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 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' Badge
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.

Primary Antibody ABIN516526
Secondary Antibody goat anti-rabbit Dye-IR800 conjugated antibody (Advansta, R-05060-250, lot 17083179)
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 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' Badge
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.

Primary Antibody ABIN516526
Secondary Antibody PE-conjugated rabbit anti-mouse immunoglobulin antiserum (Jackson ImmunoResearch, 115-116-146, lot 120701)
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.

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...
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.
Immunogen sequence: TEPPLVRVNR KETFPGVTAL FCKAHGFYPP EIYMTWMKNG EEIVQEIDYG DILPSGDGTY QAWASIELDP QSSNLYSCHV EHCGVHMVLQ VPQESETIPL
Clone 5B5
Isotype IgG1 kappa
Cross-Reactivity Human
Plasmids, Primers & others Plasmids, Primers & others MR1 products on genomics-online (e.g. as negative or positive controls)
Antigen
Alternative Name MR1 (MR1 Antibody Abstract)
Background Full Gene Name: major histocompatibility complex, class I-related
Synonyms: HLALS
Gene ID 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
Application Notes Optimal working dilution should be determined by the investigator.
Comment

Product Quality tested by: Antibody Reactive Against Recombinant Protein.

Restrictions For Research Use only
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.
Supplier Images
 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...
Did you look for something else?