核蛋白提取protocol的.docVIP

Cytoplasmic and Nuclear Protein Extraction Materials: Solution Amount Mass of Solid Other 1M HEPES pH 7.9 100ml 23.83g pH! 0.5M HEPES pH 7.6 10ml --- Dilute Above by 2X and pH 1M MgCl2 10ml 0.9522g --- 3M KCl 10ml 2.2368g --- 1M KCl 3ml --- Dilute above by 3X. Glycerol --- --- --- 0.1M CaCl2 10ml 147mg Note mg! 1M MgAc 10ml 2.145g --- 1M Sucrose 10ml 3.423g --- 250mM EDTA 50ml 4.653g pH 8.1, very tedious 100mM DTT 10ml 154.25mg Note mg! Aliquot and freeze at -20°C 100mM PMSF 10ml 174.2g Note mg! Store at -20°C. Dissolve in EtOH. Reagents: Low Salt Buffer-For 10mL200μL of 1M HEPES pH 7.92.5mL of glycerol15μL of 1M MgCl2200μL of 1M KCl8μL of 250mM EDTA100μL of 100mM DTT郃DD FRESH!50μL of 100mM PMSF郃DD FRESH!6.927mL of dH2OHigh Salt Buffer-For 10mL200μL of 1M HEPES pH 7.92.5mL of glycerol15μL of 1M MgCl22.67mL of 3M KCl8μL of 250mM EDTA100μL of NP-40100μL of 100mM DTT郃DD FRESH!50μL of 100mM PMSF郃DD FRESH!4.357mL of dH2OSucrose Buffer w/o NP-40-For 10mL3.2mL of 1M Sucrose300μL of 0.1M CaCl220μL of 1M MgAc4μL of 250mM EDTA100μL of 100mM DTT郃DD FRESH!50μL of 100mM PMSF郃DD FRESH!6.326mL of dH2OSucrose Buffer w/ NP-40-For 1mL1mL of Sucrose Buffer w/o NP-405μL of NP-40Procedure: PERFORM ALL STEPS ON ICE!1. Collect cells from 1 confluent T75 (scraping or trypsinizing, doesn抰 matter). 2. Wash cells once with ice-cold PBS and repellet.3. Resuspend cells in 1mL ice-cold PBS and transfer to an eppendorf tube. 4. Pellet cells at 200xg for 5 minutes. 5. Resuspend cells in 200mL Sucrose buffer with NP-40 by gently pipetting with a 1000mL tip, and incubate on ice for 5 minutes to lyse. 6. Pellet nuclei by centrifugation at 1500xg for 5 minutes and transfer the supernatant (cytoplasmic fraction) to a new tube. (NOTE: It抯 best to leave the last 50mL at the bottom of the tube out of your cytoplasmic fraction, this reduces the likelihood of contaminating the cytoplasmic fraction with nuclear protein.)7. Gently resuspend the nuclei in