After washing the column with B-PER wash buffer, the bound proteins were released with elution buffer (50 mM Tris, 300 mM NaCl, 200 mM imidazole, 10% [v/v] glycerol), according to a previous protocol (Shibasaki et al. [2013]).
PAS was washed three times with IP buffer and bound proteins were released by addition of SDS sample buffer and heating at 100°C for 5 min. For Coomassie blue staining of purified proteins, 2 4 μg of protein was typically loaded.
The immunocomplexes were washed 5 times with 800 µl of co-immunoprecipitation buffer, and immunoprecipitated proteins were released from the antibody-agarose-conjugate by the addition of 25 µl of glycine pH 2.5 with a 2 min incubation in ice.
Beads were finally washed three times with lysis buffer and co-immunoprecipitated proteins were released by boiling in SB (2% SDS, 20% glycerol, 100 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol).
Pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A, and the soluble nuclear proteins were released by adding 0.1 ml of buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% (w/v) glycerol, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin and 1 mM NaN3).
50 μL of 2× SDS loading buffer was added and the proteins were released for preparative SDS-PAGE by incubation for 6 min at 96 °C.
Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining.
Bound proteins were released by incubating with Laemmli sample buffer.
The purified proteins were released to 2× SDS sampling loading buffer by boiling.
Proteins were released by boiling in protein sample buffer, fractionated by SDS-PAGE and visualized by silver staining.
