Veriblot for ip dectection reagent

Wild-type retinas at P8 were collected and lysed in the RIPA buffer (ThermoFisher, #89900) with a protease inhibitor cocktail (Roche, #04693124001). Three biological replicates were performed. Cell lysate was centrifuged for 20 min at 12,000 rpm at 4°C. The super-natant was incubated with anti-ISL1 (Abcam Cat# ab20670, RRID:AB_881306) at 4°C overnight. Then, the protein A/G agarose beads (Santa Cruz Biotechnology Cat# sc-2003, RRID:AB_10201400) was added into the sample and incubated at 4°C for 4 hours. After four washes with RIPA buffer, the sample was suspended with 2x Laemmli Sample Buffer (Bio-Rad #1610737) and boiled for 10 min. Then, the sample was loaded into SDS-PAGE gel, and ran for 2 hours at 100 V. The proteins were transferred from the gel to PVDF membrane using Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Next, the membrane was blocked with the blocking buffer (5% milk in TBST) for 1 hour at RT, and incubated with anti-LHX4 (1:1000, Proteintech # 11183–1-AP) at 4°C overnight. Then, the membrane was washed three times with TBST for 5 min each, it was incubated with VeriBlot for IP Dectection Reagent (1: 1000, Abcam #ab131366) for 1 hour at RT. After the membrane was washed four times with TBST for 5 min each, detection was done with ImageQuant LAS4000 biomolecular imager (GE Healthcare).