Cells were harvested at indicated time points, lysed in reducing Laemmli buffer and proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures [33].
The column was washed with 45 ml of the same buffer, and proteins were eluted in 45 ml of a linear concentration gradient (30-0% saturation) of ammonium sulfate at a flow rate of 1.5 ml/min.
Beads were washed three times with lysis buffer, and proteins were eluted with SDS loading dye.
Sepharose was washed extensively in the lysis buffer and proteins were eluted in 500 ng/µL FLAG peptide (Sigma).
Rabbit adult fibroblasts were lysed using SDS-sample buffer and proteins were analyzed after siRNA transfection against CCT-eta and CCT-beta.
After incubation at room temperature for 30 min the reaction was stopped by adding protein loading buffer and proteins were separated on SDS-PAGE gels.
Beads were mixed with 30 µl of Laemmli blue buffer, and proteins were resolved on a 12 SDS-PAGE and transferred onto PVDF transfer membrane.
The beads were washed 3 times in lysis buffer and proteins were eluted by boiling in SDS-sample buffer containing 4% 2-mercaptoethanol (Bio-Rad).
The complex was resuspended in RIPA buffer, and proteins were eluted with SDS buffer for Western analysis to detect protein interactions among BAD, HRK and P32.
The pellet was resuspended in a 2x Laemmli sample buffer and proteins were resolved by SDS-PAGE and immunoblotted forBcl-2 and Bcl-xL as above.
HIS6SMN/GST-Gemin2 heterodimers were cleaved with Calpain1, as described above, and samples were mixed with 5X SDS sample buffer and proteins were separated by SDS-PAGE.
