Immunocruz ip wb optima e system

CHO cells transfected with RAGE and GFP-tagged PRAK were washed with PBS and lysed with 1 % CHAPS buffer (Sigma). To reduce non-specific binding, pre-clearing with protein A/G agarose (Santa Cruz Biotechnology) was performed for 1 h at 4 °C with gentle rotating. After bicinchoninic acid assay, equal protein lysates were immunoprecipitated with anti-GFP antibodies (1 μg/mL; Santa Cruz Biotechnology), incubated overnight at 4 °C with gentle rotating, and added to the beads for 1 h. The samples were washed in the lysis buffer and elution protein complex with the SDS-PAGE sample loading buffer and analyzed by western blotting as described above. Brain tissue or SH-SY5Y cells were lysed with RIPA and using ImmunoCruz™ IP/WB Optima E System (Santa Cruz Biotechnology) according to the manufacturer’s protocol with anti-RAGE antibodies (1 μg/mL; Millipore).

Co-IPs were carried out using the ImmunoCruz™ IP/WB Optima E System (Santa Cruz, UK), according to the manufacturer's instructions, with 1 mg whole cell lysate prepared by RIPA extraction for each IP. 2 μg of LRH-1 antibody or normal mouse IgG (Invitrogen) was used per IP. Following immunoprecipitation, samples were heated at 100°C for 3 min and 10 μl of immunoprecipitated complex was used for Western blotting. HRP conjugated ImmunoCruz™ E antibody was used as the secondary antibody.