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chengtao9688木虫 (小有名气)
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[交流]
【求助/交流】膜蛋白提取
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| 请问哪位大侠有成熟的膜蛋白提取方法？ |
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yunaitong
木虫 (小有名气)
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2楼2009-12-23 14:19:05
qingfengwu
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提供一份我们实验室的提取膜蛋白protocol
★ ★ ★ ★
chengtao9688(金币+4,VIP+0): 12-24 21:33
chengtao9688(金币+4,VIP+0): 12-24 21:33
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1) Plate dissociated neurons on 6 cm culture dishes for 1 hour and half at least. 2) Prepare your stimulation-solutions (37 C) as well as ice cold PBS (containing Ca/Mg) with pH 7.4 at 4 C (correct pH is very important for biotinylation reaction). 3) Additionally, need a stock solution of EZ-LINK-Sulfo-NHS-LC-Biotin from Pierce (product number 21335) of 50 mg/ml in DMSO (200x) and Glycine 1 M in water (100x) (both: storage of aliquots at -80). 4) Wash the cell once with 3 ml ECS and add 2 ml stimulation-solution with Sulfo-NHS-LC-Biotin (0.5 mg/ml) into the dish for 10 min at 37 C. 5) Continue to incubate for 30 min at 4 C (e.g. refrigerator). Move plate from time to time. Pre-cold the shaker and centrifuge. 6) Afterwards, suck off sup and add 1 M Glycine (10 mM Glycine) into the dish for 20 min at 4 C (this terminates the crosslinking reaction). 7) Suck off sup, put the dish on the ice and wash 3 times with 2 ml of ice cold PBS (+Ca/Mg). 8) Subsequently, add 350 μl of RIPA solution (PMSF 17.4 mg/ml (100x), leupetin 8μl /1ml, PEPA (1000x), Aprotonin (2000x)) for cell-lysis and collect the cells into prechilled Eppendorf-tubes, 1 hr at 4 C on shaker. 9) Spin 10 min at 14.000 rpm in cold room. 10) Take sups and transfer to pre-chilled fresh tubes (get rid of pellet which contains DNA). Take 50μl into pre-chilled fresh tubes for whole cell lysis and add 17μl 4x loading buffer. Mix and boil for 5 min in waterbath. 11) Start immunoprecipitation by adding 30 microliters of Streptavidin-Agarose (Pierce 20347) (use a yellow tip with cut off top). Note: shake the Streptavidin-Agarose before adding. 12) Rotate overhead at 4 C for at least 12 hours (or overnight). 13) Finish immunoprecipitation by spinning down your agarose-beads (10,000 rpm 4 min). Take sups into pre-chilled tubes for the cytoplasmic lysis and 17μl 4x Loading buffer into 50 μl. 14) Wash 4 times with 0.5 ml ice-cold RIPA solution (PMSF 17.4 mg/ml (100x), leupetin 8μl /1ml, PEPA (1000x), Aprotonin (2000x)), spin down at 8,000 rpm 4min and take away sups with a syringe (1 ml) each time. 15) Finally add 50 μl 1x Loading buffer for the surface lysis and boil for 5 min or 50C 20 min in waterbath together with the cytoplasmic lysis. 16) Keep the lysis at -20C and spin down the surface lysis before loading your samples onto the gel. |
3楼2009-12-23 15:13:02
liudaihualyh
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Preparation of membranes from skeletal muscles. Red and white gastrocnemius and soleus muscles from rat hindlimb were removed and trimmed of connective tissue, fat, and nerves. The muscles were then minced and homogenized on ice three times (10 s each) using a Polytron homogenizer set at 13,500 rpm in a buffer containing 20 mM HEPES, 250 mM sucrose, 1mMEDTA, 5mMbenzamidine, 1 μMaprotinin A, 1 μM pepstatin, 1 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4. The homogenate was centrifuged at 2,000 g for 10 min. The pellet, which contained mainly unhomogenized pieces of tissue, was discarded, and the supernatant was centrifuged at 9,000 g for 20 min. The 9,000-g pellet (P1) was resuspended in PBS with the standard cocktail of protease inhibitors listed above and analyzed for the presence of marker proteins by Western blotting. The supernatant was centrifuged at 180,000 g for 90 min. The 180,000-g pellet was resuspended in PBS with protease inhibitors, loaded on a 10–30% (wt/wt) continuous sucrose gradient (3–4 mg protein/5 ml gradient), and centrifuged at 48,000 rpm for 55 min in a SW-50.1 rotor. Gradients were separated into 25–30 fractions, starting from the bottom of the tube. The pellet of the sucrose-gradient centrifugation (P2) was resuspended in PBS and analyzed together with the gradient fractions. All centrifugations were performed at 4°C. |
4楼2009-12-25 23:20:31
