Ez magna rip rna binding protein immunoprecipitation kit 17 701

Proteins extracted from primary patient cells or cell lines were resolved by 7.5%, 10%, or 12% Bis-Tris polyacrylamide gels and were transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% BSA for 1 h and probed with the appropriate antibody overnight at 4 °C and then were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Membranes were visualized with an enhanced chemiluminescence detection system. Detail information of antibody was mentioned in Additional file 1: Table S5.
For RIP assays, FLAG- or HA-tagged fusion proteins were used. In the RIP experiment with FLAG-DOT1L (N- terminal) or HA-DOT1L (N- terminal) truncated mutants in 293T cells, anti-FLAG (Sigma, USA) or anti-HA (Sigma, USA) was used along with an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17-701) (Merck Millipore, Germany) according to the manufacturer’s instructions. All proteins for RIP were lysed with cell lysis buffer supplemented with Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA). Finally, all samples were suspended in 5× loading buffer and then denatured for 5 min at 100 °C, separated via SDS-PAGE, transferred to PVDF membranes, and blotted.

The RIP assay was performed using the EZ-Magna RIP RNA-binding protein immunoprecipitation kit 17-701 (Merck Millipore, Germany) according to the manufacturer’s instructions. Briefly, BGC-823 cells were collected and lysed in RIP lysis buffer with protease and RNase inhibitors. The cell lysates were incubated with magnetic beads conjugated with Ago2 antibodies or IgG (Millipore) at 4 °C overnight. Then, the beads were washed and incubated with proteinase K to remove proteins. Finally, RNA was extracted and subjected to RT-PCR and agarose gel electrophoresis analysis.

The EZ-Magna RIP RNA-binding protein immunoprecipitation kit 17-701 (Merck Millipore, Germany) was used to performed RIP assay according to the manufacturer’s product specification. Firstly, cells were collected and lysed in complete RIP lysis buffer. Then, the cell extract was incubated with RIP buffer containing magnetic beads conjugated to a human anti-Ago2 antibody (Millipore, USA) or IgG (Millipore, USA) at 4 °C overnight. Samples were incubated with proteinase K with shaking to digest proteins and the immunoprecipitated RNA was isolated and the purified RNA was subjected to real-time PCR analysis.