GADD34/PPP1R15A Rabbit Polyclonal Antibody

IHC analysis of GADD34/PPP1R15A using anti-GADD34/PPP1R15A antibody. GADD34/PPP1R15A was detected in a paraffin-embedded section of human melanom tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml rabbit anti-GADD34/PPP1R15A Antibody overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.

Western blot analysis of GADD34/PPP1R15A using anti-GADD34/PPP1R15A antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human K562 whole cell lysates, Lane 2: human HEL whole cell lysates, Lane 3: human RT4 whole cell lysates, Lane 4: human U251 whole cell lysates, Lane 5: human SIHA whole cell lysates, Lane 6: human U-87MG whole cell lysates, Lane 7: human PC-3 whole cell lysates, Lane 8: rat testis tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-GADD34/PPP1R15A antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for GADD34/PPP1R15A at approximately 100 kDa. The expected band size for GADD34/PPP1R15A is at 73 kDa.