Immunoprecipitation of Heat Shock Protein 70 (Hsp70) was performed on THP-1 cells. Antigen-antibody complexes were formed by incubating 500ug of THP-1 whole cell lysate (in 500ul volume) with 5ul of an Hsp70 monoclonal antibody (Product # MA1-10889) overnight at 4?C. The immune complexes were captured on 30ul of protein G agarose, washed extensively, and eluted with 6X Laemmli non-reducing sample buffer. Samples were resolved on an 8% SDS-PAGE gel, transferred to a PVDF membrane, and blocked with 5% milk in TBST for 1 hour at room temperature. The membrane was probed with an Hsp70 monoclonal antibody (Product # MA1-10889) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:40,000 for 1 hour at room temperature. Chemiluminescent detection was performed using ECL substrate. Data courtesy of the Innovators Program.
Western blot analysis of Heat Shock Protein 70 (Hsp70) was performed by loading 30ug of THP-1 cell lysate per well onto an SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. The membrane was probed with an Hsp70 monoclonal antibody (Product # MA1-10889) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:40,000 for 1 hour at room temperature. Chemiluminescent detection was performed using ECL substrate. Data courtesy of the Innovators Program.