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happykeren2008捐助贵宾 (正式写手)
空空道人
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[交流]
求助：大鼠肝微粒体的制备问题
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1、肝微粒体在上海有哪些大学可以制备？ 费用大概多少？ 2、肝微粒体制备的经验，有经验的虫子可否留个联系方式？ 3、很急  |
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2楼2008-08-18 10:24:30
txlfox
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3楼2008-09-14 15:05:06
zhongjie
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PREPARATION OF MICROSOMES FROM RAT LIVER Rat liver microsomes can be prepared from the following protocol, which was adapted from the original method of Omura et al. (1956), and characterized for P450 content (see Support Protocol 2). Microsomes can be prepared from other animal liver tissue either fresh or snap frozen in liquid nitrogen and stored up to 1 year at −80°C. Alternatively, liver microsomes from human, dog, mouse, rat, monkey, and other species can be purchased from a number of commercial suppliers including BD-Gentest, In Vitro Technologies, Panvera, and Xenotech. Materials Rats Homogenizing buffer (see recipe), ice cold Pyrophosphate buffer (see recipe), ice cold Microsome buffer (see recipe), ice cold 0.1 M phosphate buffer, pH 7.4 10-ml syringe and 22-G needle Ice bath Surgical scissors Hand-held blender (e.g., Bio Homogenizer) Teflon pestle (Potter type), motor driven Centrifuge tubes, ice cold Ultracentrifuge tubes Ultracentrifuge, refrigerated Additional reagents and equipment for determining protein concentration by standard methods (APPENDIX 3A) Prepare tissue 1. Sacrifice animals by cervical dislocation, decapitation, or CO2 asphyxiation and exsanguinate. Avoid use of chemical anesthetics such as halothane, barbiturates, and chloroform, which may affect activity and distribution of metabolic enzymes. Overnight fasting of animals before sacrificing is advisable to reduce the glycogen content of the liver, which can interfere with microsome isolation. 2. Remove the liver rapidly and place in ice-cold homogenizing buffer in an ice bath. Perfuse the liver by inserting a 10-ml syringe with a 22-G needle into the liver vasculature and injecting buffer until the effluent is clear and colorless (－10 ml buffer for a rat liver). It is essential to keep the tissue at <4°C at all times to prevent loss of enzyme activity. Liver vasculature can be identified as several small openings. The liver color will blanch immediately upon successful perfusion. 3. Remove excess moisture by blotting on paper towels and weigh the tissue. The liver tissue may be stored up to 1 year at –80°C prior to microsome preparation. Large livers should be cut into smaller portions and snap frozen in liquid nitrogen. Thaw on ice after covering the frozen tissue with cold homogenizing buffer. 4. Add 3 vol of ice-cold homogenizing buffer and mince the liver into small pieces with surgical scissors. Divide the tissue into portions of －4 g each. 5. Further break apart the tissue on ice with a hand-held blender (e.g., Bio Homogenizer) for 3 to 5 sec. Take care to prevent frothing, which will cause denaturation of enzymes. Because dog and monkey livers are a tougher tissue than rat and mouse livers, they may require more extensive homogenization. 6. Homogenize, on ice, using a motor-driven Teflon pestle (Potter type) with ∼10 strokes. Place homogenate into an ice-cold labeled 1.5-ml centrifuge tube. Isolate microsomes 7. Centrifuge the homogenate 15 min at 12,500 × g, 4°C. The supernatant is the S9 homogenate fraction; enzyme activities are maintained for years when these homogenates are stored at –70°C. 8. Carefully decant the supernatant into a 1.5-ml ultracentrifuge tube and discard the pellet. Balance pairs of tubes to within 0.01 g using ice-cold homogenizing buffer, pH 7.4. 9. Ultracentrifuge the supernatant 70 min at 105,000 × g, 4°C. For example, a Beckman L8-80 ultracentrifuge equipped with a Ti 70 rotor with a 6.57-cm radius is run at 38,000 rpm. 10. Decant the supernatant and resuspend the pellet in 8 ml ice-cold pyrophosphate buffer. Homogenize the pellet using the hand-held blender (e.g., Bio Homogenizer) for 3 to 5 sec. 11. Rebalance the tubes and ultracentrifuge 45 min at 105,000 × g, 4°C. 12. Decant the supernatant, add 6 ml ice-cold microsome buffer and resuspend the pellet with a hand-held blender. Further homogenize the pellet with the Potter-type Teflon pestle and transfer into a clean tube. Wash the Teflon pestle with 2 ml ice-cold microsome buffer and combine. 13. Determine the protein concentration of a small aliquot by standard methods (APPENDIX 3A). Adjust to the desired protein concentration (usually 10 to 20 mg/ml) with microsome buffer. 14. Dispense 0.5-ml aliquots into labeled tubes and store at −70° to −80°C. Enzyme activities are maintained for years when stored at −70°C. Avoid repeated freezethaw cycles. Microsomes from other tissue (e.g., kidney, lung, brain, intestine, and skin) can also be isolated by this method, although increased homogenization may be required. [ Last edited by zhongjie on 2008-9-16 at 18:21 ] |
4楼2008-09-16 18:18:25
zhongjie
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DETERMINATION OF CYTOCHROME P450 (CYP) CONTENT IN MICROSOMES CYP contains a heme iron (Fe2+) that gives a characteristic absorption spectra at 450 nm when complexed with CO. The quantity of CYP is determined by measuring the absorbance difference between 450 and 490 nm and using the extinction coefficient (ε)= 91 mM−1cm−1 (Omura and Sato, 1964). A yield of 0.4 to 1.0 nmol CYP/mg protein from untreated livers can be expected. Materials Microsomal preparation (see Support Protocol 1) 0.2 M phosphate buffer, pH 7.4 Crystalline dithionite (Na2S2O4 or sodium hydrosulfite) Carbon monoxide (CO) in a tank with a two-stage valve Spectrophotometric cuvettes with 1-cm light path (quartz or plastic) UV/VIS spectrophotometer, preferably a dual-beam instrument 1. Dilute microsomes to 1 mg/ml protein in 0.2 M phosphate buffer, pH 7.4, and mix by gently vortexing or by inversion. Approximately 4 ml of microsome solution is needed for standard cuvettes. 2. Dispense 2 ml of the microsomal preparation into matched sample and reference cuvettes and record a baseline spectrum between 400 and 500 nm. 3. Add a few crystals of dithionite (<5 mg) to both cuvettes and mix gently to dissolve. Dithionite acts as a reducing agent to ensure that all heme iron is in a reduced state. Reduced CYP is unstable, so minimize the time between dithionite addition and CO treatment. 4. Bubble CO into the sample cuvette only for ∼30 sec at a rate of 1 bubble/sec. An excessively high flow rate of CO will cause frothing and protein denaturation. 5. Re-scan and record the spectrum from 400 to 500 nm. The spectrum should exhibit an absorption maximum at 450 nm. A prominent peak at 420 nm indicates the presence of hemoglobin or inactivated CYP. 6. Calculate CYP content in the diluted sample as follows: CYP concentration (mM) = A/ε where A = A450 – A490, the absorbance difference between 450 and 490 nm and ε = 91 mM–1cm–1, the extinction coefficient (450 - 490 nm) for CYP. 7. Multiply the CYP concentration (μM) in the diluted sample (from step 6) by the dilution factor to determine the CYP content in the original undiluted sample. REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX. Homogenizing buffer 0.1 M Tris⋅Cl, pH 7.4 (APPENDIX 2A) 10 mM EDTA 1.15% KCl (150 mM) Store up to 6 months at 4°C Microsome buffer 0.05 M Tris⋅Cl, pH 7.5 (APPENDIX 2A) 10 mM EDTA 20% glycerol Store up to 6 months at 4°C NADPH regenerating system (10×) 30 mM glucose-6-phosphate (pH 7.4) 4 U/ml glucose-6-phosphate dehydrogenase 10 mM NADP (pH 7.4) 30 mM MgCl2 Prepare immediately before use Pyrophosphate buffer 0.1 M Na pyrophosphate, pH 7.4 10 mM EDTA Store up to 6 months at 4°C Testosterone, 0.5 mM Prepare 1.45 mg/ml in ethanol (5 mM, store up to 6 months at –20°C); dilute 20 μl with 180 μl of 0.1 M phosphate buffer, pH 7.4. |
5楼2008-09-16 18:33:00