1. Growing Cells and Addition of Exogenous Antigens

1. Prepare bOVA using a biotin-protein labeling kit following the manufacturer's protocol.
   NOTE: Ordinarily, bOVA contains 2 M biotin per 1 M OVA on average.
2. Grow DC2.4 cells in RPMI-1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5), and 10% fetal calf serum (hereafter RPMI) at 37 °C in 5% CO₂ in a humidified incubator (hereafter without mention, cells are incubated at this condition). It is also possible to use DMEM supplemented with 10% fetal calf serum, 3.7 g/L NaHCO₃ (hereafter DMEM) in place of RPMI.
3. A day before purification, split the cells into RPMI to 1 x 10⁵ cells/mL in a tissue culture plate. Avoid keeping the cells at a confluent state. DC2.4 cells can highly incorporate exogenous antigens until a semi-confluent state, but rapidly lose the ability after reaching an over-confluent state.
4. Before the DC2.4 cells are semi-confluent, add 250 µg/mL of bOVA for 1 x 10⁶ cells/mL and incubate for 2 – 4 h.
   NOTE: During downstream experiments, all buffers and reagents are kept at 4 °C unless otherwise indicated.

2. Preparation of Microsomes

1. Harvest the DC2.4 cells by gentle pipetting from the tissue plate into a new 50 mL conical tube.
2. Centrifuge at 1,000 x g for 5 min, 4 °C and carefully remove the medium with bOVA by aspiration.
3. Wash the DC2.4 cells twice with 40 mL of PBS buffer (1.37 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄) by centrifugation at 1,000 x g for 5 min, 4 °C and discard the supernatant each time by aspiration.
4. Resuspend the pellet in step 2.3 in 1 – 2 mL (1/20-1/10 volume of culture medium) of homogenization medium (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES-NaOH [pH 7.4]) with 1/1,000 volume of protease inhibitor cocktails and transfer the resuspended DC2.4 cells into an ice-cold Dounce homogenizer.
5. Disrupt the resuspended DC2.4 cells gently by 10 – 20 strokes with the ice-cold Dounce homogenizer.
   NOTE: During the disruption step, the Dounce homogenizer is cooled on ice.
6. Transfer the cell-disrupted suspension to a new 15 mL conical tube.
7. Add 8 – 9 mL homogenization medium to 10 mL total and centrifuge the conical tube for 10 min at 2,000 x g and 4 °C.
8. Transfer the supernatant to a new 15-mL conical tube to remove unbroken cells and nuclei, and centrifuge the conical tube again for 10 min at 2,000 x g and 4 °C.
9. Transfer the supernatant into a new ultracentrifugation tube and centrifuge for 45 min at 100,000 x g and 4 °C.
10. Aspirate the supernatant and resuspend the pellet carefully in 1 – 2 mL of homogenization medium with 1/1,000 volume of protease inhibitor cocktails by pipetting the solution up and down several times to make a microsome fraction for downstream experiments.
11. Transfer the microsome fraction into a new 5-mL round bottom tube.
    NOTE: After this step, it is possible to enrich objective microsomes by iodixanol density gradient centrifugation according to the manufacturer's protocol, to remove non-specific microsomes. The peak fractions for exogenous antigens are collected and then subjected to the next step of purification (step 3).

3. Purification of Microsomes with bOVA Undergoing ERAD

1. Add 1/100 volume of fresh SA-magnetic beads to the microsome fraction of step 2.11 in the 5 mL round bottom tube.
   NOTE: Before this step, it is possible to pre-clear the microsome fraction by control-magnetic beads to reduce contaminations of non-specific microsomes.
2. Gently mix well and rotate the round bottom tube slowly for 30 min at 4 °C.
3. Add 2-3 mL of homogenization medium to 4 mL total into the round bottom tube and gently mix well.
4. Place the round bottom tube on a magnetic stand and incubate for 10 min at 4 °C. Since the beads bound to the vesicles are attracted to the magnet, brown micro-beads will gradually accumulate to the tube wall closest to the magnet.
5. With the tube remaining in the magnetic stand, carefully discard the supernatants by aspiration to remove the unbound vesicles.
6. Wash the magnetic beads bound to the vesicles twice with 5 mL of homogenization medium by the magnetic stand for 10 min at 4 °C and discard the supernatant each time by carefully aspiration.
   NOTE: Without the magnetic stand, purification by centrifugations at 2,000 x g for 10 min, 4 °C is also available.
7. Resuspend the magnetic beads bound to the vesicles carefully in 100 µL homogenization medium by pipetting the solution up and down several times.
8. Transfer the resuspended vesicles into a new microtube as the purified microsome for downstream experiments.
   NOTE: Typically, 50 µL microsome fraction containing 5 – 20 µg proteins from 1 x 10⁷ cells can be isolated.

4. Analysis of the Purified Microsomes

1. Resuspend the magnetic beads bound to the vesicles from step 3.8 by 100 – 200 µL of TNE buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 M EDTA, 1% Nonidet P-40) with 1/1,000 volume of protease inhibitor cocktails instead of the homogenization medium.
2. Transfer the lysate from step 4.1 to a new 1.5-mL microtube.
3. Determine the protein concentration of step 4.2 by using a BCA assay kit per the manufacturer's protocol.
   NOTE: Typically, 5 – 10 µL lysate from step 4.2 is enough to determine the protein concentration.
4. Transfer the lysate from step 4.2 (2 µg of protein for silver staining and 10 µg of protein for Western blotting) into new microtubes.
5. Put the microtubes on a heat block at 95 °C and boil proteins in 1x SDS gel-loading buffer (100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) for 5 min.
6. Centrifuge the microtubes for 10 min at 21,500 x g and 4 °C. Collect the supernatants into new microtubes to remove the insoluble fractions.
7. Analyze the resolved proteins in step 4.6 (2 µg) by standard SD-PAGE.
8. Visualize the protein bands by the silver staining using silver staining kit following the manufacturer's protocol.
9. Analyze the resolved proteins in step 4.6 (10 µg) by standard SD-PAGE and Western-blotting. Use the reagent (SA-HRP) per the manufacturer's protocol and visualize by fluorography.

5. In Vitro Reconstitution of ERAD Ubiquitination of bOVA Using Purified Microsomes

1. Transfer the purified microsomes (5 – 10 µg of protein) from step 3.8 with a 50% volume of reticulocyte lysate (RL), in 1x reaction buffer (50 mM Tris pH 7.4, 3 mM ATP, 0.5 mM MgCl₂), and 0.2 pM Flag-tagged ubiquitin in a new microtube. The final volume of this experiment is 20 – 40 µL.
2. Incubate the microtube for 2 h at 37 °C.
   NOTE: If the amount of ubiquitinated bOVA in step 5.12 is too small to detect by Western blotting, add 10 µM of MG132 or 2 µM of lactacystin to inhibit the processing of the ubiquitinated bOVA by proteasomes.
3. Stop the reaction by placing the microtube on ice.
4. Resolve the microsomes by adding 500 µL TNE buffer with 1/1,000 volume of protease inhibitor cocktails.
5. Centrifuge the microtube for 10 min at 21,500 x g and 4 °C. Collect the supernatants into a new microtube to remove the insoluble fractions, which contain ubiquitinated bOVA in DC2.4 before the in vitro ubiquitination assay.
6. Transfer the supernatant to a new microtube and add 1/100 volume of new SA-magnetic beads (usually 5 µL for 500 µL of supernatants).
7. Gently mix well and rotate the microtube slowly for 30 min at 4 °C.
8. Centrifuge the microtube for 10 min at 21,500 x g and 4 °C. Discard the supernatant by aspiration to recover the bOVA and ubiquitinated bOVA bound with the SA-magnetic beads.
9. Wash twice the collected SA-magnetic beads with 1 mL of TNE buffer by centrifugation for 10 min at 21,500 x g and 4 °C. Discard the supernatant each time by aspiration.
10. Boil the SA-magnetic beads in 1x SDS gel loading buffer for 5 min at 95 °C on a heat-block to resolve the purified proteins by the SA-magnetic beads.
11. Centrifuge the microtube for 10 min at 21,500 x g and 4 °C. Collect the supernatants into a new microtube to remove the insoluble fractions.
12. Analyze the SA-magnetic beads bound to the proteins by standard SD-PAGE and Western-blotting. The use of antibodies (anti-Flag, anti-multi-Ub, and anti-mouse IgG-HRP) and the reagent (SA-HRP) is per the manufacturer's protocol and visualized by fluorography.