Cell extracts were prepared using lysis buffer (1% NP-40, 150 mM NaCl, 100 mM Hepes, 5 mM Na4P2O7, 5 mM NaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 mg/l aprotinin, 10 mg/l leupeptin, PMSF).
For GST pull-down assay, the lysates were incubated with Glutathione-Sepharose 4B beads (GE Healthcare) at 4 °C 4 h on rotation. After three washes with lysis buffer, the bound proteins were released in SDS loading buffer.
For the Flag pull-down assay, the lysates were first incubated with anti-Flag M2 affinity gel (Sigma) at 4 °C overnight with rotation. After incubation, complexes were washed three times with lysis buffer, and the bead-conjugated proteins were released with SDS loading buffer.
For GST pull-down assay, the lysates were incubated with Glutathione-Sepharose 4B beads (GE Healthcare) at 4 °C 4 h on rotation. After three washes with lysis buffer, the bound proteins were released in SDS loading buffer.
For the Flag pull-down assay, the lysates were first incubated with anti-Flag M2 affinity gel (Sigma) at 4 °C overnight with rotation. After incubation, complexes were washed three times with lysis buffer, and the bead-conjugated proteins were released with SDS loading buffer.
Free full text: Click here
