Gfp trap magnetic beads

Ovaries from ~170 GFP-Panx, GFP-Nxf2 and control flies (3–5 days old) were dissected in ice-cold PBS and lysed in 300 μl of CoIP Lysis Buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, 0.2% NP-40 supplemented with complete protease inhibitors [Roche]) and homogenized using a motorized pestle. Lysates were cleared for 5 min at 16000 g and the residual pellet re-extracted with the same procedure. GFP-tagged proteins were immunoprecipitated by incubation with 30 µl of GFP-Trap magnetic beads (Chromotek) for 3 hr at 4°C on a tube rotator. The beads were washed 6x with Lysis Buffer and 2x with 100 mM Ammonium Bicarbonate, before TMT-labelling followed by quantitative Mass Spectrometry. TMT chemical isobaric labeling were performed as described (Papachristou et al., 2018 (link)).

Stable hTERT-RPE1 cellines expressing GFP or GFP WDFY2 were seeded in 10 cm dishes up to 80% confluence and then lysed in lysis buffer containing, 50 mM TRIS, 150 M NaCl, 0.25% Triton X100, 1 mM DTT, 50 µM ZnCl₂, 5 mM NaPPi, 20 mM NaF, 1× of phosphatase inhibitor 3 (S/T), phosphatase inhibitor 2 (Y), and protease inhibitor mix. GFP-trap magnetic beads (ChromoTek) were added to the lysate and incubated rotating at 4° for 4 h.

To prepare cell extract, 5 g of frozen cell pellets were ground using a SPEX SamplePrep LLC 6850 freezer/mill. Subsequently, and prior digestion, Cbk1-GFP was immunoprecipitated on GFP-Trap Magnetic beads (ChromoTek, gtd-20). Immunoprecipitated proteins were washed 4 times with 200 mM ammonium bicarbonate (ABC) and resuspended in 6 M Urea/200 mM ABC. Then, the beads were reduced and alkylated with 10 mM dithiothreitol (DTT) and 57 mM chloroacetamide (CAA) for 1 h and 30 min, respectively, and digested with 1 μg of trypsin for 16 h at 30 °C. The tryptic peptides were recovered by pulling-down the beads. The supernatant was recovered to a new, clean tube and the digestion was stopped by adding 10% formic acid (FA). The peptides were then desalted using a PolyLC C18 pipette tip, dried in a speedvac, and stored to −80 °C.

Flp-in HeLa cells were treated with thymidine and doxycycline for 24 h, followed by a treatment with nocodazole and doxycycline for 16 h. Mitotic cells were isolated by mitotic shakeoff and incubated with media containing nocodazole and doxycycline with or without calyculin A (50 nM; LC Laboratories) for 20 min. Cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na₃VO₄, 5 mM β-glycerophosphate, 25 mM NaF, 10 nM calyculin A, and complete protease inhibitor containing EDTA; Roche) on ice for 20 min. The lysate was incubated with GFP-Trap magnetic beads (from ChromoTek) for 2 h at 4°C on a rotating wheel in wash buffer (same as lysis buffer, but without Triton X-100) at a 3:2 ratio of wash buffer:lysate. The beads were washed three times with wash buffer, and the sample was eluted according to the protocol from ChromoTek.

For immunoprecipitation, 150 pairs of testes were lysed in 0.5 ml HEPES based buffer [50mM HEPES pH 7.5, 250mM NaCl, 0.1% Nonidet P40, 0.2% Triton X-100, supplied with cOmplete protease inhibitor mixture (Roche)]. In Klp10A co-immunoprecipitation (co-IP) experiments, the cleared lysates were incubated with rabbit anti-Klp10A serum (1:200, [37 (link)]) for 4 hours, extended by 2 extra hours after supplement with Protein A Dynabeads (Life Technologies). The beads were washed four times with lysis buffer and proteins were eluted with SDS-PAGE protein sample buffer. For co-IP experiments with GFP tagged proteins, the cleared lysates were incubated with GFP-Trap magnetic beads (Chromotek) according to manufacturer's instructions. To detect precipitated proteins on western-blots, the following primary antibodies were combined with horseradish peroxidase conjugated secondary antibodies (Abcam): guinea-pig anti-Klp10A (1:10,000, [37 (link)]), rabbit anti-GFP (1:3000, ab290, Abcam), rabbit anti-Vasa d-26 (1:3000, Santa Cruz Biotechnology), rabbit anti-Piwi (1:3000 [76 (link)]), rabbit anti-Aub (1:3000 [76 (link)]), and mouse anti-Ago3 (1:3000 [12 (link)]).