Anti gfp antibody

TE8 cells were plated at 2.5 × 10⁶ per 10‐cm dish and transiently transfected with either pCMV6‐AC‐GFP‐SIM2 or no insert of pCMV6‐neo (OriGene Technologies) by using Lipofectamine 2000 reagent (Invitrogen). Cells were treated with 5 μmol/L proteasome inhibitor, MG‐132 (Sigma‐Aldrich, St Louis, MO, USA) for 18 hours and, 48 hours after transfection, cells were collected and lysed in lysis buffer (20 mmol/L Tris‐HCl, 20% glycerol, 300 mmol/L KCl, 5 mmol/L 2‐mercaptoethanol, 1 mmol/L Pefabloc (Roche, Basel, Switzerland), and 25 μmol/L MG‐132. Immunoprecipitation was conducted using Immunoprecipitation Kit‐Dynabeads Protein G (Life Technologies, Rockville, MD, USA). Cell lysates were incubated with anti‐GFP antibody (OriGene Technologies) or control mouse IgG at 4°C overnight, and then incubated with 50 μL Dynabeads at 4°C for 4 hours. The Dynabeads‐antibody‐antigen complex was washed and resuspended in SDS sample buffer, then incubated at 95°C for 5 minutes. Eluted proteins were fractionated by SDS‐PAGE and detected by western blot.