Before starting
Cool the microcentrifuge to 4˚C and prepare a bucket with crushed ice to cool tubes.
Steps
Preparation of coupling reaction of anti-APOE antibodies to Dynabeads (~1 h).
1. Pool two tubes with 0.1 mg of anti-pan-APOE IgG and centrifuge for 10 min at 16,000 × g, 4˚C to pellet any potential antibody aggregates. After centrifugation, leave on ice.
2. Weight out 40 mg of Dynabeads in a 1.5-ml microcentrifuge tube.
3. Add 0.8 ml of C1 buffer from the Dynabeads Antibody Coupling Kit to the Dynabeads in the 1.5-ml microcentrifuge tube and resuspend by gentle vortexing or pipetting up and down.
4. Divide the Dynabead suspension equally over two 2-ml microcentrifuge tubes (0.4 ml/tube).
5. Place the two 2-ml microcentrifuge tubes with the Dynabead suspension in a magnetic tube rack for 1 min and remove supernatant.
6. Add 1 ml C1 buffer minus the volume of 0.1 mg anti-pan-APOE IgG to each of the two 2-ml microcentrifuge tubes with Dynabead pellet. The anti-pan-APOE IgG concentration is usually around 1 mg/ml. Please check the label and calculate the volume of 0.1 mg anti-pan-APOE IgG to determine the volume of C1 buffer to add (0.9 ml of C1 if the IgG concentration is 1 mg/ml).
7. Resuspend the Dynabead pellets in the two 2-ml microcentrifuge tubes by gentle vortexing or pipetting up and down.
8. Add 0.1 mg of anti-pan-APOE IgG in solution (step 1) to each of the two 2-ml microcentrifuge tubes with resuspended Dynabeads. The total volume should now be 1 ml in each tube.
9. Add 1 ml of C2 buffer from the Dynabeads Antibody Coupling Kit to each of the two 2-ml microcentrifuge tubes with Dynabead/antibody mixture.
Overnight covalent coupling of anti-APOE antibodies to Dynabeads.
10. Incubate the two 2-ml microcentrifuge tubes with Dynabead/antibody mixture overnight, placed in a rotating tube rack at 37˚C.
Buffer changes and washes of anti-APOE antibody-coupled Dynabeads (~1.5 h).
11. The next day, centrifuge the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads briefly in a microcentrifuge (pulse to 400 × g) to remove any Dynabeads touching the lid.
12. Place the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads in a magnetic tube rack for 1 min and remove supernatant.
13. Add 0.9 ml of HB buffer from the Dynabeads Antibody Coupling Kit to each of the two 2-ml microcentrifuge tubes with antibody-coupled Dynabead pellet and resuspend the pellets by gentle vortexing or pipetting up and down.
14. After a quick spin in the microcentrifuge (see step 11), place the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads in a magnetic tube rack for 1 min and remove supernatant.
15. Add 0.9 ml of LB buffer from the Dynabeads Antibody Coupling Kit to each of the two 2-ml microcentrifuge tubes with antibody-coupled Dynabead pellet and resuspend the pellets by gentle vortexing or pipetting up and down.
16. After a quick spin in the microcentrifuge, place the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads in a magnetic tube rack for 1 min and remove supernatant.
17. Add 0.9 ml of SB buffer from the Dynabeads Antibody Coupling Kit to each of the two 2-ml microcentrifuge tubes with antibody-coupled Dynabead pellet and resuspend the pellets by gentle vortexing or pipetting up and down.
18. After a quick spin in the microcentrifuge, place the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads in a magnetic tube rack for 1 min and remove supernatant.
19. Repeat steps 17 and 18.
20. Add 0.9 ml of SB buffer from the Dynabeads Antibody Coupling Kit to each of the two 2-ml microcentrifuge tubes with antibody-coupled Dynabead pellet and resuspend the pellets by gentle vortexing or pipetting up and down.
21. Place the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads in a tube rotator and mix for 15 min at room temperature.
22. After a quick spin in the microcentrifuge, place the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads in a magnetic tube rack for 1 min and remove supernatant.
At this stage, the antibody-coupled Dynabeads can be stored, resuspended in SB buffer (0.033–0.1 ml of SB buffer per mg of beads) at 4˚C until use or used directly for immunoprecipitation of APOE-lipoprotein particles.
Prior to the immunoprecipitation, the antibody-coupled Dynabeads need to be repeatedly washed with PBS to remove any traces of detergents present in the SB buffer that would interfere with the co-immunoprecipitation of phospholipids (1–1.5 h).
23. Wash the two 2-ml microcentrifuge tubes with antibody-coupled Dynabeads six times with PBS as follows:
a. Add 0.9 ml of PBS to each tube and resuspend the pellets by gentle vortexing or pipetting up and down.
b. Place the tubes in a tube rotator and mix for 5 min at room temperature.
c. After a quick spin in the microcentrifuge, place the tubes in a magnetic tube rack for 1 min and remove supernatant.
24. After the last wash, add 2 ml of PBS to each of the two 2-ml microcentrifuge tubes with antibody-coupled Dynabead pellet and resuspend the pellets by gentle vortexing or pipetting up and down.
Immunoprecipitation of APOE-lipoprotein particles with anti-APOE antibody-coupled Dynabeads (~1.5 h).
25. Transfer 0.5 ml of the resuspended antibody-coupled Dynabeads from the two 2-ml microcentrifuge tubes to eight 1.5-ml microcentrifuge tubes.
26. Place the eight 1.5-ml microcentrifuge tubes with antibody-coupled Dynabeads in a magnetic tube rack for 1 min and remove supernatant.
27. Thaw out eight CSF samples protected from light to preserve the integrity of the phospholipids and pipette 0.4 ml of each CSF sample into eight new 1.5-ml microcentrifuge tubes labelled with the code of the CSF sample.
28. Centrifuge the eight 1.5-ml tubes with CSF samples for 10 min at 16,000 × g, 4˚C to pellet any potential cell debris.
29. During the centrifugation, label the eight 1.5-ml microcentrifuge tubes with antibody-coupled Dynabead pellet (step 26) with the codes of the CSF samples.
30. Add 0.4 ml of supernatant from the eight CSF samples to the eight 1.5-ml microcentrifuge tubes with antibody-coupled Dynabead pellet. Resuspend the pellets by gently vortexing or pipetting up and down.
31. Place the eight 1.5-ml microcentrifuge tubes with antibody-coupled Dynabeads/CSF mixture in a tube rotator and mix for 1 h at room temperature, protected from light (put, e.g., a cardboard box over the tube rotator).
Washing and preparation of the anti-APOE antibody-coupled Dynabead/CSF immunoprecipitate for mass spectrometric analyses (~1.5 h).
32. After a quick spin in the microcentrifuge, place the eight 1.5-ml microcentrifuge tubes with the antibody-coupled Dynabead/CSF mixture in a magnetic tube rack for 1 min.
33. Transfer supernatants (= first wash) from the eight 1.5-ml microcentrifuge tubes with the antibody-coupled Dynabead/CSF mixture to new labelled 1.5-ml microcentrifuge tubes and store at -80˚C. These supernatants can be used to analyze the phospholipids or proteins that have not immunoprecipitated.
34. Wash the eight 1.5-ml microcentrifuge tubes with antibody-coupled Dynabead/CSF immunoprecipitate pellet (step 32) six times with PBS as follows:
a. Add 0.9 ml of PBS to each tube and resuspend the pellets by gentle vortexing or pipetting up and down.
b. Place the tubes in a tube rotator and mix for 5 min at room temperature protected from light.
c. After a quick spin in the microcentrifuge, place the tubes in a magnetic tube rack for 1 min and remove supernatant.
35. After the final wash, centrifuge the eight 1.5-ml microcentrifuge tubes with the antibody-coupled Dynabead/CSF immunoprecipitate pellets for 1 min at 16,000 × g in a microcentrifuge and carefully remove all traces of fluid.
36. Store the eight 1.5-ml tubes with the antibody-coupled Dynabead/CSF immunoprecipitate at -80˚C.
37. Perform mass spectrometric analyses within days of the immunoprecipitation to preserve the integrity of the samples. Transport samples protected from light on dry ice.