MR1 antibody (AA 201-300)

Catalog No. ABIN516526

This independently validated Mouse Monoclonal antibody specifically detects MR1 in ELISA, WB and IF. It exhibits reactivity toward Human.

Quick Overview for MR1 antibody (AA 201-300) (ABIN516526)

Target: MR1 (Major Histocompatibility Complex, Class I-Related (MR1))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This MR1 antibody is un-conjugated

Application: ELISA, Western Blotting (WB), Immunofluorescence (IF)

Clone: 5B5

Product Details anti-MR1 Antibody

Binding Specificity: AA 201-300

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.

Isotype: IgG1

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (ICC)

Validation #101752 (Immunocytochemistry)

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.

Validation Images

Human MR1-expressing HEK293 cells were stained with MR1 antibody ABIN516526 and a FITC-conjugated secondary antibody (green, A). For nuclear staining, cells were stained with Hoechst (blue, B). C shows both channels merged.

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.

Independent Validation (IP)

Validation #102826 (Immunoprecipitation)

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.

Validation Images

Lysates from human MR1-expressing HEK293 cells were immunoprecipitated by antibodies specific for MR1 (ABIN2665876, ABIN516526, ABIN1537116) or the respective isotype controls (mouse IgG2a, mouse, IgG1, rabbit IgG). Immunoprecipitants were resolved by SDS-PAGE gel followed by Western blotting analysis using MR1 antibody ABIN1537116.

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.

Independent Validation (FACS)

Validation #102827 (Flow Cytometry)

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.

Validation Images

Human MR1-expressing HEK293 cells (MR1) and vector control cells (Vc) were stained with MR1 antibodies ABIN516526 or ABIN2665876 followed by PE-conjugated anti-mouse secondary antibody. For total MR1 staining, cells were permeabilized with 0.1% saponin and stained with primary and secondary antibodies. Cells were analyzed by flow cytometry.

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.

Handling

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 Details for MR1

Target: MR1 (Major Histocompatibility Complex, Class I-Related (MR1))

Alternative Name: MR1

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