Protein g sepharose beads

The protein G Sepharose beads (193259, Abcam) were activated by rinsing three times with 1000 µL of lysis buffer and centrifuging at 150× g. Total cell lysates obtained from the previous method were pre-cleared with 50 µL protein G Sepharose beads and incubated at 4 °C for 1 h, followed by gentle mixing using a rotating mixer. The pre-cleared lysate was recovered by centrifugation at 1200× g for 5 min and precipitated with the protein of interest antibody (Ab). The 5 µL of c-Myc Ab was added into pre-cleared lysate and incubated overnight at 4°C with moderate mixing on a rotating mixer. The immune protein complex was recovered by centrifugation at 1200× g for 5 min at 4 °C, then gently resuspended with cold lysis solution 3 times. The immune protein complex was recovered by centrifugation at 1200× g for 5 min at 4 °C, and the protein concentration was quantified by Bradford assay using a Bio-Rad protein assay kit. The immune protein complex was probed by Western blot analysis.

Brain homogenates prepared from cortex of mice or lysates of N2a-APPswe cells were diluted (1:1) with binding buffer, 0.05 M sodium borate, pH 8.0, 0.15 M NaCl, and then incubated with 10 μl of Protein G-Sepharose beads (Biovision, Mountain View, CA) (pre-washed with the binding buffer) with gentle agitation at 4 °C for 1 h to reduce non-specific binding. After centrifugation at 10,000 × g for 10 min, the supernatants were used for immunoprecipitation (IP). Samples were incubated at room temperature for 1 h with the IP-antibodies, mouse anti-PrP mAb SAF-32 at a ratio of 1:100 (v/v). After addition of 10 μl pre-washed Protein G-Sepharose beads, the mixture was rolled at 4 °C overnight. Antigen-antibody-Protein G-sepharose complexes were collected by centrifugation at 14,000 r.p.m. for 10 min and washed 3 times with 1 ml of the binding buffer. Proteins were eluted with 60 μl of elution buffer, 0.1 M citric acid, pH 2.75. The eluate was mixed with 20 μl of 4 × SDS-loading buffer without β-ME and adjusted to pH 8.0 with 1 μl of 10 M NaOH, and then boiled for 5 min. Samples (30 μl each) were separated using 12.5% SDS-PAGE to detect PrP or 6% and 16.5% discontinuous tricine-tris SDS-PAGE gels for Aβ detection. Following transfer to PVDF membranes, proteins and peptides were probed with human anti-PrP antibody D13 (1:1000) or rabbit anti-Aβ₄₂ antibody PA3-16761 (1:1000).

Protein extraction and western blots were performed according to standard procedures (Wodarz, 2008 (link)). For Co-IPs, transiently transfected S2R+ cells were lysed in 1 ml cold Co-IP lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% Triton X-100 with protease inhibitors) by pipetting up and down in a 1000 µl micropipette and incubation on ice for 30 min. The lysates were centrifuged and the supernatant was transferred into fresh tubes and pre-cleared with Protein G Sepharose beads (BioVision, Milpitas, USA) for 1 h on a rotator at 4°C. After pre-clearing, 20 μl of each sample were kept as input control. Lysate corresponding to 1 mg of total protein was incubated with 2 µg of mouse anti GFP (Invitrogen, A11120) for 1 h at 4°C. Next, 20 µl Protein G Sepharose beads were added to the lysates and incubated overnight at 4°C on a rotator. Beads were washed three times with 1 ml Co-IP buffer. After removal of all remaining liquid with a syringe, the beads were boiled with 2× SDS-loading buffer and stored at −20°C or used directly for SDS-PAGE and western blot.
Antibodies used for western blot were rabbit anti GFP, 1:2000 (A11122, Invitrogen) and mouse anti c-myc (9E10), 1:200 (DSHB).