Clone epr7130 b

Total cell lysates were prepared in RIPA Buffer (Pierce) supplemented with HALT Protease and Phosphatase Inhibitor (Pierce). Lysates were pelleted at 12 000 × g for 20 min and the supernatant was collected. Protein lysates were quantified using a Bicinchoninic Acid Assay (Pierce). Protein lysates were mixed with NuPAGE LDS Sample Buffer (Life Technologies), loaded onto a 4–12% Bis-Tris gradient gel (Life Technologies), and subjected to electrophoresis in MES buffer (Life Technologies). The proteins were transferred to a nitrocellulose membrane using an iBlot gel transfer device (Life Technologies). The nitrocellulose membrane was blocked with a 5% non-fat milk solution prepared in TBS-Tween (Cell Signaling). Primary antibodies to TSG101 (1:2000, clone EPR7130(B), Millipore) and β- Actin (1:10000, AC-15, Santa Cruz) were incubated overnight at 4°C with rocking in 2% non-fat milk/TBS-Tween solution. Membranes were extensively washed and incubated with secondary antibody (1:5000, Pierce). Membranes were developed with Super Signal West Pico Chemiluminescent Substrate (Pierce) and exposed to film.