INSTRUCTIONS FOR RNA ISOLATION (15 to 30°C) 1. HOMOGENIZATION Cell Monolayer – Lyse cells directly in a culture dish by adding 1 ml of TRIZOL to a 3.5 cm diameter dish (10 cm2), & passing the cell lysate several times through a pipette. 2. PHASE SEPARATION • Incubate the homogenized samples for 5 min. at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. • Add 0.2 ml of chloroform per 1 ml of TRIZOL. Shake tubes vigorously by hand for 15 seconds & incubate them at 15 to 30°C for 2 to 3 min. • Centrifuge the samples at no more than 2,000 × g for 15 min. at 2 to 8°C. 3. RNA PRECIPITATION • Transfer the aqueous phase to a fresh tube, & save the organic phase if isolation of protein is desired. INSTRUCTIONS FOR PROTEIN ISOLATION (15 to 30°C) 1. DNA PRECIPITATION • Remove the remaining aqueous phase overlying the interphase, & precipitate the DNA from the interphase & organic phase with ethanol. • Add 0.3 ml of 100% ethanol per 1 ml of TRIZOL & mix samples by inversion. • Store the samples at 15 to 30°C for 2-3 min. & sediment DNA by centrifugation at no more than 2,000 × g for 5 min. at 2 to 8°C. • Remove the phenol-ethanol supernatant, & if desired, save it for protein isolation. 1. PROTEIN PRECIPITATION • Precipitate proteins from the phenol-ethanol supernatant with 1.5 ml of isopropyl alcohol per 1 ml of TRIZOL. • Store samples for 10 min. at 15 to 30°C, & sediment the protein precipitate at 12,000 × g for 10 min. at 2 to 8°C. 2. PROTEIN WASH • Remove the supernatant & wash the protein pellet 3 times in a solution containing 0.3 M guanidine hydrochloride in 95% ethanol. Add 2 ml of wash solution per 1 ml of TRIZOL. • During each wash cycle, store the protein pellet in the wash solution for 20 min. at 15 to 30°C & centrifuge at 7,500 × g for 5 min. at 2 to 8°C. • After the final wash, vortex the protein pellet in 2 ml of ethanol. • Store the protein pellet in ethanol for 20 min. at 15 to 30°C & centrifuge at 7,500 × g for 5 min. at 2 to 8°C. 3. REDISSOLVING THE PROTEIN PELLET • Dissolve it in Digestion Buffer. Complete dissolution of the protein pellet may require incubating the sample at 50°C. Sediment any insoluble material by centrifugation at 10,000 × g for 10 min. at 2 to 8°C, & transfer the supernatant to a fresh tube. The sample may be stored at -5 to -20°C for future use. Do Bradford assay to get amt of protein (assume you have between 15-50mg/ml protein for dilutions) Dilute into DIGESTION buffer to make 4mg/ml protein Heat to 90deg C, 5 min Cool to 37 degC Add trypsin solution ("shake" dry trypsin to bottom of the bottle, add 20uL of ImM sterile HCL to dissolve trypsin add all of it to 500ul of 4mg/ml protein to give 1:100 w/w of trypsin to protein) Digest for 18hrs, 37degC (If there is still precipitate then centrifuge off the precipitate and add another l0ug of trypsin.) Centrifuge in eppendorf, 5 min @ 10,000g (invisible pellet is insoluble material) Dilute 1/20 into Mass Spec buffer C and analyze