For IP experiments in S2R+ cultured cells, protocol was followed as described [60 (link)] with minor changes: 2 mg of the protein lysates was incubated for 2 h with 10 μl of either Myc-Trap or GFP-Trap beads (Chromotek). To determine the dependence of interactions on RNA, 50 U of RNaseT1 (ThermoFisher) were added to the respective IP. To ensure the activity of RNase T1, lysates were incubated 10 min at RT prior to the incubation of lysate with antibody.
For IP experiments in ovaries, 150 μl of wet ovaries from 3–5 day old flies expressing Venus-Mkrn1 was homogenized on ice in 2 ml of cold IP buffer (1 X PBS, 0.4% Triton X-100, 1 mM MgCl2, 5% glycerol), containing protease inhibitors and PMSF. The extracts were diluted to 1.5 mg protein/ml. Each extract (0.66 ml) was mixed with 24 μg of anti-pAbp Fab antibody (Smibert lab, [61 (link)]), 17 μg of α-eIF4G rabbit antibody, or 15 μl of rabbit anti-α−Tubulin antibody (Abcam). When present, 100 μg RNase A (Qiagen) was added to the samples. Samples were incubated with rotation at 4°C overnight, then mixed with 30 μl of protein A agarose beads (wet volume, Invitrogen) and incubated with rotation at RT for 1.5 h. The beads were washed three times with IP buffer. Bound material on the beads was eluted by boiling for 2 min in 40 μl of SDS loading buffer. 20 μl of the eluted sample, together with input samples, was used for western blot.
For IP experiments in ovaries, 150 μl of wet ovaries from 3–5 day old flies expressing Venus-Mkrn1 was homogenized on ice in 2 ml of cold IP buffer (1 X PBS, 0.4% Triton X-100, 1 mM MgCl2, 5% glycerol), containing protease inhibitors and PMSF. The extracts were diluted to 1.5 mg protein/ml. Each extract (0.66 ml) was mixed with 24 μg of anti-pAbp Fab antibody (Smibert lab, [61 (link)]), 17 μg of α-eIF4G rabbit antibody, or 15 μl of rabbit anti-α−Tubulin antibody (Abcam). When present, 100 μg RNase A (Qiagen) was added to the samples. Samples were incubated with rotation at 4°C overnight, then mixed with 30 μl of protein A agarose beads (wet volume, Invitrogen) and incubated with rotation at RT for 1.5 h. The beads were washed three times with IP buffer. Bound material on the beads was eluted by boiling for 2 min in 40 μl of SDS loading buffer. 20 μl of the eluted sample, together with input samples, was used for western blot.
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