Table 2. Cell Membrane Isolation from Nucleus-Containing Cells and Intracellular Organellea.
| cell type | hypotonic buffer (ice cold) | disruption technique (ice cold) | method of isolation (4 °C) | ref |
|---|---|---|---|---|
| Intracellular Organelle: Mitochondria | ||||
| isolation of mitochondria from mouse liver | 210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl,1 mM EDTA | Kinematic Polytron PT-2000 homogenizer (power setting 7 for 15 strokes) | 2000g, 10 min | (91) |
| 7000g, 10 min | ||||
| followed by sucrose density gradient ultrafiltration | ||||
| top: 15 mL of 1.0 M sucrose solution | ||||
| bottom: 15 mL of 1.5 M sucrose solution | ||||
| 60,000g, 20 min | ||||
| mitochondria- interface between the two sucrose layers | ||||
| isolation of membrane from mitochondria | ultapure water (20 min) followed by addition of 1.4 M sucrose solution (5 min) | Kinematic Polytron PT-2000 homogenizer (power setting 7 and 30 strokes) | differential centrifugation | |
| 12,000g, 10 min | ||||
| 100,000g, 30 min | ||||
| Immune Cells | ||||
| neutrophils | 0.5% (w/v) BSA, 75 mM sucrose, 225 mM mannitol, 0.5 mM EDTA, 30 mM Tris-HCl, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (50–100 passes) | differential centrifugation | (55) |
| 800g, 10 min | ||||
| 10,000g, 20 min | ||||
| 100,000g, 60 min | ||||
| 75 mM sucrose, 225 mM d- mannitol, 0.5 mM EGTA, 30 mM Tris-HCl, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (66) | |
| 20,000g, 25 min | ||||
| 100,000g, 35 min | ||||
| NK-92 | 10 mM Tris-HCl, 10 mM MgCl2, 1 mM KCl, 25 mM sucrose, 2 mM PMSF, 200 μg/mL trypsin chymotrypsin inhibitor, 10 μg/mL DNase, 10 μg/mL RNase | homogenized, 5 min (20 s pulse, 30 s in between pulses) | discontinuous sucrose gradient ultracentrifugation | (67, 102) |
| 30%, 40%, and 55% sucrose in 0.85% saline | ||||
| cell membrane: interface between 30% and 40% | ||||
| cell lysis buffer, protease inhibitor cocktail | water bath sonication, 20–30 min | differential centrifugation | (150) | |
| 3500g, 10 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 50 min | ||||
| mouse natural killer cells | cell lysis buffer, protease inhibitor cocktail | water bath sonication, 20–30 min | differential centrifugation | (150) |
| 3500 g, 10 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 50 min | ||||
| human cytotoxic T-lymphocyte cells | 10 mM Tris-HCl, 1 mM KCl, 1 mM MgCl2, phosphatase inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | discontinuous sucrose gradient ultracentrifugation | (98) |
| 30%, 40%, and 55% | ||||
| cell membrane: interface between 30% and 40% | ||||
| RAW264.7 | 10 mM Tris-HCl, 1 mM MgCl2 | Mini-extruder without a polycarbonate membrane (20 times) | homogenate mixed with 1 M sucrose to a final concentration of 0.25 M sucrose | (129) |
| centrifugations | ||||
| 2000g, 10 min | ||||
| 3000g, 30 min | ||||
| 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | ||
| 3200g, 5 min | ||||
| 20,000g, 30 min | ||||
| 80,000g, 1.5 h | ||||
| 1 mmol/L NaHCO3, 0.2 mmol/L EDTA, 1 mmol/L PMSF | repeatedly grinding (20 times) | differential centrifugation | (69) | |
| 3200g, 5 min | ||||
| 100,000g, 30 min | ||||
| THP-1 | 10 mM Tris-HCl, 10 mM MgCl2, 1 mM KCl, 25 mM sucrose, 2 mM PMSF, 200 μg/mL trypsin chymotrypsin inhibitor, 10 μg/mL DNase, 10 μg/mL RNase | Dounce homogenizer, tight-fitting pestle (20–30 passes) | discontinuous sucrose gradient ultracentrifugation | (63) |
| 30%, 40%, and 55% sucrose in 0.85% saline | ||||
| cell membrane: interface between 30% and 40% | ||||
| J774 | 10 mM Tris-HCl, 10 mM MgCl2, 1 mM KCl, 25 mM sucrose, 2 mM PMSF, 200 μg/mL trypsin chymotrypsin inhibitor, 10 μg/mL DNase, 10 μg/mL RNase | Dounce homogenizer, tight-fitting pestle (20–30 passes) | discontinuous sucrose density gradient ultracentrifugation | (63) |
| 30%, 40%, and 55% sucrose in 0.85% saline | ||||
| cell membrane: interface between 30% and 40% | ||||
| 75 mM sucrose, 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (130) | |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 35 min | ||||
| dendritic cells | 25 mM sucrose, 10 mM Tris-HCl, 1 mM MgCl2, 1 mM KCl, 2 mM PMSF, protease and phosphatase inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (25 passes) | differential centrifugation | (106) |
| 800g, 5 min | ||||
| 21,000g, 10 min | ||||
| Cancer Cells | ||||
| HeLa | membrane and cytosol protein extraction kit and PMSF | 3 freeze–thaw cycles | differential centrifugation | (219) |
| 700g, 10 min | ||||
| 14,000g, 30 min | ||||
| MCF-7 | HEPES B buffer: 2.38 g/L Hepes, 0.476 g/L MgCl2, 0.292 g/L EDTA, 0.154 g/L DTT, 0.746 g/L KCl, proteinase inhibitor cocktail | IKA T10 basic homogenizer (three times, four-speed position) | discontinuous sucrose density gradient ultracentrifugation | (198) |
| 30%, 40%, and 55% | ||||
| cell membrane: interface between 30% and 40% | ||||
| 0.5% (w/v) BSA, 75 mM sucrose, 225 mM mannitol, 0.5 mM EDTA, 30 mM Tris-HCl, protease inhibitor cocktail | sonication | differential centrifugation | (175) | |
| 800g, 10 min | ||||
| 10,000g, 20 min | ||||
| 100,000g, 60 min | ||||
| 4T1 | 10 mM Tris, 10 mM MgCl2, protease inhibitor cocktail | homogenizer (22,000 rpm for 1 min) | differential centrifugation | (220, 221, and 72) |
| 500g, 10 min | ||||
| 10,000g, 20 min | ||||
| 100,000g, 60 min | ||||
| B16-F10 | 30 mM Tris-HCl, 75.9 mM sucrose, 225 mM d-mannitol, phosphatase inhibitor, protease inhibitor cocktail | Kinematica Polytron PT 10/35 probe homogenizer (70% power, 15 passes) | differential centrifugation | (222) |
| 10,000g, 25 min | ||||
| 150,000g, 35 min | ||||
| 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | pipetting thoroughly | differential centrifugation | (214) | |
| 3200g, 5 min | ||||
| 21,000g, 25 min | ||||
| 45,000g, 5 min | ||||
| 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (92) | |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 35 min | ||||
| LL/2, CMT64.OVA, MB49, A549, SKOV-3 | 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | pipetting thoroughly | differential centrifugation | (214) |
| 3200g, 5 min | ||||
| 21,000g, 25 min | ||||
| 45,000g, 5 min | ||||
| K562 | 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (92, 103) |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 35 min | ||||
| MDA-MB 435 | 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (92, 223) |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 35 min–1.5 h | ||||
| patient-derived tumor cell | 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (105) |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 80,000g, 1.5 h | ||||
| DU145, CAL27, HCT116 | 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (223) |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 80,000g, 1.5 h | ||||
| U87 | 225 mM mannitol, 75 mM sucrose, mM EDTA, 30 mM Tris-HCl, protease inhibitor cocktail | motor-driven homogenizer with an overhead stirrer (2000 rpm); tissue grinder set with a smooth PTFE pestle (Wheaton Potter-Elvehjem) | differential centrifugation | (224) |
| 1000g, 5 min | ||||
| 7000g, 10 min | ||||
| 100,000g, 30 min | ||||
| followed with discontinuous sucrose gradient ultracentrifugation | ||||
| 2 M, 1.6 M, 1.2 M sucrose buffer | ||||
| cell membrane: 1 M density | ||||
| U-251 MG | distilled water | high-pressure homogenizer (6000 psi/412.5 bar, 10 passages) | differential centrifugation | (67) |
| 10,000g, 10 min | ||||
| 60,000g, 90 min | ||||
| LNCaP-AI | Mem-PERPlus membrane protein extraction kit | stirring for 10 min | 700g, 5 min | (225) |
| 16,000g, 15 min | ||||
| H22 | 18 mM Tris-HCl, 9 mM KCl, 1.5 mM MgCl2, protease inhibitor cocktail | Freeze–thawing, bath ultrasonic cleaner (42 kHz, 100 W) | differential centrifugation | (226) |
| 3000g, 6 min | ||||
| 20,000g, 25 min | ||||
| 120,000g, 1 h | ||||
| Stem Cells | ||||
| neural stem cells | 10 mM Tris, 10 mM MgCl2, protease inhibitor cocktail | freeze–thawing | differential centrifugation | (188) |
| 7000g, 10 min | ||||
| 13,000g, 60 min | ||||
| human adipose-derived stem cells | 10 mM Tris, 10 mM MgCl2, protease inhibitor cocktail | IKA T10 basic homogenizer (22,000 rpm for 1 min) | 6000 g, 15 min | (227) |
| Miscellaneous | ||||
| MIN6 | 20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (96) |
| 20,000g, 20 min | ||||
| 100,000g, 45 min | ||||
| C6/36 | 20 mM Tris HCl, 10 mM KCl, 2 mM MgCl2, protease inhibitor cocktail | Dounce homogenizer, tight-fitting pestle (20 passes) | differential centrifugation | (228) |
| 3200g, 5 min | ||||
| 20,000g, 25 min | ||||
| 100,000g, 35 min | ||||
a
Abbreviations/cell lines: min, minutes, h, hour; BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Tris-HCl, tris(hydroxymethyl) aminomethane (THAM) hydrochloride; MgCl2, magnesium chloride; KCl, potassium chloride; PMSF, phenylmethylsulfonyl fluoride; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DTT, dithiothreitol; DNase, deoxyribonuclease; RNase, ribonuclease; NK-92, human natural killer cell line; RAW 264.7, murine macrophage cell line; J774, murine macrophage cell line; THP-1, human acute monocytic leukemia cell line; HeLa, human cervical cancer cell line; MCF-7, human breast cancer cell line; 4T1, human breast cancer cell line; B16-F10, murine melanoma cell line; LL/2, murine lewis lung carcinoma cell line; CMT64.OVA, lung carcinoma cell line expressed ovalbumin; MB49, murine bladder carcinoma cell line; A549, human nonsmall cell lung cancer cell line; SKOV-3, ovarian cancer cell line; CAL27, human squamous cancer cell line; H22, murine hepatocarcinoma cell line; K562, human myelogenous leukemia cell line; MDA-MB-435, human breast carcinoma cell line; DU145, human prostate cancer cell line; CAL27, human squamous cancer cell line; U-251 MG, glioblastoma multiforme cell line; HCT116, human colorectal cancer cell line; U87, human primary glioblastoma cell line; MIN6, pancreatic β-cell line; C6/36, Aedes albopictus mosquito medium host cell line; LNCaP-AI, prostate cancer cell line.