Protocol: Cell Lysate Preparation REAGENTS & BUFFERS + RIPA buffer 50 mM Tris-HCI, p74 25 mlof 1M NP-40 5 ml Na-deoxycholate 259 ‘SDS 059 NaCl 15 mi of 5M EDTA 2 mi of 05M NaF 1.059 Final 500 ml Store at 4°C. Add the protease/phosphatase inhibitors (see below table)* immediately prior use, + Lysis 250 buffer 50 mM Tris-HCl, pH 7.4 25 mlof 1M 05% NP-40) 25 mi 250 mM NaCl 25 mi of SM omM EDTA 5 mi of 5M 50 mM NaF 1.059 Final 500 ml Store at 4°C. Add the protease/phosphatase inhibitors (see below table)* immediately prior use. * Common Protease and Phosphatase Inhibitors Name Stock Conc Stocks Working Cone Phenylmethylsulfonyl fluoride (PMSF) 4100 mM stock solution in isopropanol; store -20°C in aliquots_'0°"™! 100% tm Benzamidine-HCI Store -20°C in aliquots, 100 mM in methanol 100 mM 100K tmM Leupeptin (Leu) 41 maim 1 5 ual store frozen in aliquots, 1 mg/ml in H20 mahal ox 9.6 vail ‘Aprotinin (Apr) im im store frozen in aliquots, 11 mgim| in H20 {moll 100% 98 baie Pepstatin (Pep) mgm mgm store frozen in aliquots, 11 mgiml in methanol 1 maim! 100% ‘maim! ‘Sodium Fluoride (NaF) already in som RIPA = ‘Sodium Orthovanadate (Na3VO4)" see the following protocol to prepare and activate Na-Or- 200 mM, 100% 0.2mm thovanadate stock tel 877.436.3830 or 949.559.1000 «ax 949.200.2088 - wow. genatox.com 8 GeneTex; Inc.Protocol: Cell Lysate Preparation ** Activation of Sodium Orthovanadate 1 2, 7 Sodium orthovanadate should be activated for maximal inhibition of protein phosphotyrosyl-phosphatases. Prepare a 200 mM solution of sodium orthovanadate, ‘Adjust the pH to 10.0 using either 1 N NaOH or 1 N HCl. The starting pH of the sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 the solution will be yellow. Boil the solution unti it turns colorless (approximately 10 minutes). Cool to RT. Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0. Store the activated sodium orthovanadate as aliquots at -20°C. This procedure depolymerizes the vanadate, converting it into a more potent inhibitor of protein tyrosine phosphatases. METHOD |. Monolayer Colls 1 ‘The following steps should be performed on ice or at 4°C using pre-cold buffers. Remove culture medium and rinse a subconfluent, 100 mm cell culture plate with PBS twice Detach cells with a rubber policeman in 1 ml cold PBS and transfer cell suspension into a 1.5 ml microcentsi- fuge tube, Pellet cells by centrifuging at 3,000 rpm for § min. Remove the supernatant. ‘Suspend the pellet with 1.0 ml cold RIPA buffer (or other appropriate buffer) with freshly added (Protease Inhibi- tors) and/or (Phosphatase Inhibitors). ‘Allow the tube to stand on ice for 30 min, vortex every 10 min. Centrifuge the resulting mixture at 14,000 x g for 15 min at 4°C. This separates the total protein (supernatant) from the cellular debris (pellet). Transfer supernatant to a new tube for further analysis. The call lysate can be frozen at this point for long-term storage at -80°C. Il, Suspension Cells 1 8 GeneTex; Inc. tel 877.436.3839 or 949.553.1900 ox 949.308.2688 - wor ge Collect approximately 5.0 x 107 cells by low-speed centrifugation at RT for § min. Carefully remove culture medium, Wash the pellet with PBS at RT, and collect by low-speed centrifugation. Carefully remove supematant, Add 1.0 ml of pre-cold RIPA buffer (or other appropriate buffer) with freshly added (Protease Inhibitors) and! or (Phosphatase Inhibitors). Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 minProtocol: Cell Lysate Preparation Further disrupt and homogenize cells by passing through a 21-gauge needle, dounce homogenization or soni: cation, taking care not to raise the temperature of the lysate. (Optional: Add 10 ul of 10 mg/ml PMSF stock) Incubate 30 min on ice. Transfer to microcentrifuge tube(s) and centrifuge at 10,000 x g for 10 min at 4°C. The supernatant fluid is the total cell lysate. Transfer the supematant to a new microfuge tube and discard the pellet. IIL Tissue Samples 4 tol 877.436.2829 or 949.553.1900 fax 949.209.2688 worw.ge Weigh tissue and dice into very small pieces using a clean razor blade, Frozen tissue can be sliced very thinly and thawed in RIPA buffer containing (Protease Inhibitors) and/or (Phosphatase Inhibitors). Use 3m! of pre- cold RIPA buffer per gram of tissue. Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator, while maintaining tem- peratures at 4°C throughout all procedures. (Optional: Add 30 ul of 10 mg/ml PMSF stock per gram of tissue.) Incubate on ice for 30 min. Transfer to microcentrifuge tubes, centrifuge at 10,000 x g for 10 min at 4°C. Remove supernatant and centri- fuge again. The supernatant fluid is the total cell lysate. A longer centrifugation may be necessary to obtain 2 clear lysate. 8 GeneTex; Inc. a