MR1 antibody (C-Term)

Catalog No. ABIN1537116

Product Details anti-MR1 Antibody

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

Binding Specificity: AA 312-341, C-Term

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This MR1 antibody is un-conjugated

Application: Western Blotting (WB)

Purification: This antibody is purified through a protein A column, followed by peptide affinity purification.

Immunogen: This MR1 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 312-341 amino acids from the C-terminal region of human MR1.

Clone: RB37199

Isotype: IgG

Application Details

Application Notes: WB: 1:1000

Restrictions: For Research Use only

Independent Validation (ICC)

Validation #101753 (Immunocytochemistry)

by: Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine

No.: #101753

Date: 02/20/2018

Antigen: MR1

Lot Number: SA111213CH

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 ABIN1537116 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 ABIN1537116 and a Texas Red-conjugated secondary antibody (red, A). For nuclear staining, cells were stained with Hoechst (blue, B). C shows both channels merged.

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.

Lysates from human MR1-expressing HEK293 cells (MR1) and vector control cells (Vc) were resolved on a 10% SDS-PAGE gel for Western blotting analysis using antibodies specific for MR1 (ABIN1537116, upper panel) and beta-actin (ABIN962807, lower panel).

Full Methods

Primary Antibody: ABIN1537116

Secondary Antibody: Texas Red-conjugated donkey anti-rabbit immunoglobulin antiserum (Jackson ImmunoResearch, 711-076-152, lot 66576)

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 cells in sterile glass-bottom 35-mm dishes coated with collagen (MatTek, P35GCol-1.5-14-C). Let cells grow to 50-80% confluency.
• Wash cells d with 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 atRT.
• Incubate cells with primary rabbit anti-MR1 antibody (antibodies-online, ABIN1537116, lot SA111213CH) 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.
• Incubate cells with Texas Red-conjugated donkey anti-rabbit immunoglobulin antiserum (Jackson ImmunoResearch, 711-076-152, lot 66576) diluted 1:50 in dilution buffer for 1h at RT.
• Wash cells 3x with PBS.
• To stain the nucleus, cells were immersed in PBS-containing Hoechst diluted 1:2000 in PBS 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 ABIN1537116 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 #102828 (Immunoprecipitation)

by: Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine

No.: #102828

Date: 02/20/2018

Antigen: MR1

Lot Number: SA111213CH

Method validated: Immunoprecipitation

Positive Control: HEK293 cells transfected with human MR1 cDNA

Negative Control: HEK293 cells transfected with plasmid vector only

Notes:

Passed. ABIN1537116 immunoprecipitates human MR1 overexpressed by HEK293 cells.

Validation Images

Human MR1-expressing HEK293 cells were stained with MR1 antibody ABIN1537116 and a Texas Red-conjugated secondary antibody (red, A). For nuclear staining, cells were stained with Hoechst (blue, B). C shows both channels merged.

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.

Lysates from human MR1-expressing HEK293 cells (MR1) and vector control cells (Vc) were resolved on a 10% SDS-PAGE gel for Western blotting analysis using antibodies specific for MR1 (ABIN1537116, upper panel) and beta-actin (ABIN962807, lower panel).

Full Methods

Primary Antibody: ABIN1537116

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 ABIN1537116, but not the isotype control, immunoprecipitates with human MR1 overexpressed by HEK293 cells.

Independent Validation (WB)

Validation #102829 (Western Blotting)

by: Dr. Randy Brutkiewicz Laboratory, Department of Microbiology and Immunology, Indiana University School of Medicine

No.: #102829

Date: 02/20/2018

Antigen: MR1

Lot Number: SA111213CH

Method validated: Western Blotting

Positive Control: HEK293 cells transfected with human MR1 cDNA

Negative Control: HEK293 cells transfected with plasmid vector only

Notes:

Passed. ABIN1537116 recognizes human MR1 overexpressed by HEK293 cells in a western blot.

Validation Images

Human MR1-expressing HEK293 cells were stained with MR1 antibody ABIN1537116 and a Texas Red-conjugated secondary antibody (red, A). For nuclear staining, cells were stained with Hoechst (blue, B). C shows both channels merged.

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.

Lysates from human MR1-expressing HEK293 cells (MR1) and vector control cells (Vc) were resolved on a 10% SDS-PAGE gel for Western blotting analysis using antibodies specific for MR1 (ABIN1537116, upper panel) and beta-actin (ABIN962807, lower panel).

Full Methods

Primary Antibody: ABIN1537116

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).
• Denature 200µg total protein for 5min at 95°C in 20µl 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.
  • loading control rabbit anti beta-actin (antibodies-online, ABIN962807) diluted 1:500 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 10min with PBS/0.05% Tween-20.
• Reveal protein bands using an Odyssey imaging system (LI-COR Biosciences).

Experimental Notes:

The human MR1 antibody ABIN1537116 reveals a protein of the expected molecular weight of MR1 in lysates of human MR1-expressing HEK293 cells. The protein bands is only visible in the positive but not the negative controls.

Handling

Format: Liquid

Buffer: Purified polyclonal antibody supplied in PBS with 0.09 % (W/V) sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: MR1 Antibody (C-term) can be refrigerated at 2-8 °C for up to 6 months. For long term storage, keep at -20 °C.

Expiry Date: 6 months

Target Details for MR1

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: MR1 has antigen presentation function. Involved in the development and expansion of a small population of T cells expressing an invariant T cell receptor alpha chain called mucosal-associated invariant T cells (MAIT). MAIT cells are preferentially located in the gut lamina propria and therfore may be involed in monitoring commensal flora or serve as a distress signal. Expression and MAIT cell recognition seem to be ligand-dependent.

Molecular Weight: 39366

Gene ID: 3140

NCBI Accession: NP_001181928, NP_001181929, NP_001181964, NP_001522

UniProt: Q95460

Pathways: Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Cancer Immune Checkpoints