3 3 diamiobenzidine tetrahydrochloride dab

Cell homogenates for the DAB-induced density shift were homogenized and cleared as described above except that 8–12 monolayers per condition were used instead of 2–3 and samples were homogenized in three batches to minimize clogging of the homogenizer. Additionally, monolayers were incubated with 10 μg/ml of the indicated antibody at either 4°C for 1 hr or 37°C for 30 min. The cleared lysate was loaded on top of a 10%/32% sucrose step gradient (1 ml of each step) in a thin-wall Ultra-Clear 13×51mm ultracentrifuge tube and centrifuged for 2 hr at 37,500 rpm in an SW55 Ti rotor. Crude membranes (~300 μl) were recovered from the 10%/32% interface. 250 μl of crude membrane was mixed with 1.25 ml of 1.2 mg/ml 3,3′-diamiobenzidine tetrahydrochloride (DAB, Sigma) in 6 mmol/L imidazole pH 6, 50 mmol/L NaCl. The DAB solution was passed through a 0.2 μm syringe filter before mixing with the crude membranes. 5 μl of 5% hydrogen peroxide (from a 30% w/v stock, Sigma) was added to the DAB/membrane mixture and rotated end-over-end for 30 min at room temperature in the dark. 1.25 ml of the sample was then loaded onto a 3.75 ml 20–50% continuous sucrose gradient in a thin-wall Ultra-Clear 13×51mm ultracentrifuge tube and centrifuged for 18 hr at 37,500 rpm in an SW55 Ti rotor. 250 μl fractions were collected from the bottom. Proteins were recovered by TCA precipitation.