- 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).