Pierce classic magnetic co ip kit

Co-immunoprecipitation was performed using Pierce^TM Classic Magnetic Co-IP kit (catalog #88804; Thermo Fisher). In brief, U87 cells were lysed by adding ice-cold IP lysis/wash buffer. To prepare immune complex, 500 μg total cell lysate was incubated with SLC7A11, ALXOE3 antibody, or rabbit IgG at 4 °C overnight. The immune complex was incubated with pre-washed pierce protein A/G magnetic beads at room temperature for an hour, followed by low-pH elution and immunoblotting analysis of ALOXE3 and SLC7A11.

We applied co-immunoprecipitation (Co-IP) to verify whether there is an interaction between the two proteins using Pierce^TM classic magnetic Co-IP kit (Thermo Fisher, USA). Ice-cold IP Lysis Buffer was added in EC cells for protein lysis and extraction. The antibody against DUSP1 (#sc-373841, Santa Cruz, USA) was then added to the sample to form an immune complex. The complex was incubated overnight and then washed to remove non-bound material. Finally, low-pH elution buffer dissociated the bound immune complex from the Protein A/G.

The Pierce Classic Magnetic Co-IP Kit (Thermo, USA) combined with Western blot was utilized to detect the effect of LINC00022 over-expression on ubiquitination of p21 in ESCC cells. In brief, cells were broken up with ice-cold IP lysis buffer to obtain the supernatant containing total proteins. The cell lysates were incubated with antibodies in a tube overnight at 4 °C to form the immunoprecipitation complex. Then the pre-treated protein A/G beads were added to each tube and incubated at room temperature for 1 h. Finally, the supernatant containing the target antigen collected by the magnetic stand was used for Western blot analysis.