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老鼠肝脏细胞溶质的制备,鼠肝细胞溶质的制备

发布时间:2019-08-27 07:14编辑:生命科学浏览(133)

    核心提示:Cells:We grow our CHOs with MEM[[alpha]] 10% Bovine Calf Serum and penn/strep/glutamine. For

    核心提示:Solutions and Reagents Freshly removed or flash frozen rat liver PBS Homogenization Buffer HomogeSolutions and Reagents

    核心提示:These protocols should yield enough cytosol and organelles for 1-200 MT/Organelle motility assays.Solutions and Reage

    核心提示:These protocols should yield enough cytosol and organelles for 1-200 MT/Organelle motility assays.Solutions and Reagent

    Cells:

    We grow our CHOs with MEM[[alpha]] 10% Bovine Calf Serum and penn/strep/glutamine. For a prep it is best to grow twenty large plates and the cells should be grown to overconfluence - till they start piling up on each other

    • Freshly removed or flash frozen rat liver
    • PBS
    • Homogenization Buffer
    • Homogenization Buffer containing 0.5 mM mM MgGTP
    • 2.3 M sucrose
    • Bradford reagent and protein standards
    • PM Buffer containing 0.25 M sucrose.

    These protocols should yield enough cytosol and organelles for 1-200 MT/Organelle motility assays.

    These protocols should yield enough cytosol and organelles for 1-200 MT/Organelle motility assays.Solutions and Reagents

    Protocol Rationale:

    The protocol is identical to Tim's published in Vol 134 of Methods in Enzymology. The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen in liq N2.

    Equipment

    Solutions and Reagents

    • Freshly removed or flash frozen rat liver
    • PBS
    • Homogenization Buffer
    • Homogenization Buffer containing 0.5 mM mM MgGTP
    • 2.3 M sucrose
    • Bradford reagent and protein standards
    • PM Buffer containing 0.25 M sucrose.

    What you need:

    Equipment:

    • refractometer
    • Sorvall with cold HB-4
    • Ultra with cold SW27
    • Some sort of gradient fractionator with a fraction collector
    • 16 30 ml corex tubes

    Solutions:

    A lot of these solutions are w/w; Tim says that to make these weigh out sucrose and then add buffer till weight is 100g.

    Wash & Lysis:

    PE: 10 mM PIPES, 1mM EDTA, 8 mM BME
    Make a 50X stock and pH to 7.2 with KOH

    LB: 1mM Tris-HCl, 8 mM BME
    Make Tris as 2M stock and pH to 8.0 with HCl

    LB 0.5% NP-40 :

    PBS: 130 mM NaCl, 2 mM KCl. 8 mM Na2HPO4, 2 mM KH2PO4
    Make a 10X stock

    Night before:

    Make 600 ml of

    • 1X PBS
    • 0.1X PBS
    • 0.1X PBS, 8% ultrapure sucrose
    • 8% ultrapure sucrose
    • LB : add 280 ul BME before use
    • LB 0.5% NP-40 : add 280 ul BME before use

    and put all these buffers in coldroom

    Sucrose Gradients :

    Use ultrapure sucrose

    20% and 62.5% sucrose in 1x PE 0.1% TX-100. Make 100 grams of each as follows:
    Weigh out sucrose and then on the balance add 1X PE 0.1% TX-100 till weight is 100 grams.

    Just before pouring gradients add 28 ul BME/50 g

    Pour gradients night before or during drug treatment . I find it easiest to pour night before and store in cold.

    To pour gradients - first put a 5 ml heavy sucrose pad and then pour gradient on top of that. For the prep outlined below I pour 2 gradients in SW27 tubes:
    4 ml heavy sucrose pad .

    16 ml gradient.

    The centrosomes are very close to the bottom of this gradient. The pad eliminates them from entering the curve of the tube and also gives a little leeway in setting up the fractionation.

    Ficoll cushion:

    20% Ficoll in 1X PE 0.1% NP-40

    1. Make up PE 0.1% NP-40 .
    2. Weigh out 10g Ficoll.
    3. Add PE 0.1 % NP-40 till total weight = 50 grams.
    4. Stir at RT for several hours to dissolve and store cold.
    5. Before use, add 28 ul BME
    • 20 ml glass homogenizer equipped with Teflon pestle
    • Homogenizer or Drill press
    • Superspeed centrifuge with the equivalent of Sorvall SS-34 rotors
    • 50 ml polycarbonate centrifuge tubes
    • Ultracentrifuge with the equivalents of Beckman 50.2Ti and SW-28 rotors
    • 31.5 ml thick-walled polycarbonate ultracentrifuge tubes with screw caps
    • 25 ml Ultraclear ultracentrifuge tubes
    • Freshly removed or flash frozen rat liver
    • PBS
    • Homogenization Buffer
    • Homogenization Buffer containing 0.5 mM mM MgGTP
    • 2.3 M sucrose
    • Bradford reagent and protein standards
    • PM Buffer containing 0.25 M sucrose.

    Equipment

    Protocol:

    1. Warm up 300 ml of CHO medium to 37 deg.C and add cytochalasin B and nocodazole
    2. Make sure all buffers are in coldroom, there is rocker in the coldroom and there is a good aspirator in the coldroom. Hook up a sawed off pipet to the aspirator and make sure there is a LARGE trap
    3. Add medium with drugs to 10 plates of cells. Add medium with drugs to the other 10 plates 45' later.
    4. After 90' in drug medium process first 10 plates: bring to coldroom and wash with the following buffers:

      1X PBS

      0.1X PBS, 8% sucrose

      8% sucrose

      LB

      and then pipet on
      LB 0.5% NP-40

      . After adding the LB 0.5% NP-40 transfer the plate onto a rocker in the coldroom.

    5. After 10' pipet off the lysate into a 30 ml corex tube. Add 1/50 vol of 50X PE

    6. Spin tubes in HB-4 for 3' at 3000 rpm at 4 deg.C
    7. Transfer supe to a fresh corex tube and underlay with 2 ml of Ficoll cushion .
    8. Spin at 12,700 rpm for 15 at 4 deg.C for 15'. As soon as spin is started, process the next set of plates through steps 4-7.
    9. Aspirate supe till approx. 2 ml above cushion and then collect interface with a pasteur - see Tim's protocol. Collect ~2 ml / tube and pool. Check Ficoll concentration by refractometry and dilute to 10% or lower - necessary to make sure it layers onto the sucrose gradient and doesn't sink.
    10. Finish collecting interfaces from second set of plates and then pool all collected interfaces, ensure Ficoll is < 10% and load onto 1 gradient. Spin 1 hr - 1hr 30', SW 27 at 2 deg.C.
    11. Fractionate gradient from bottom - 0.3 - 0.5 ml fractions and read sucrose concentration by refractometry. Assay fractions between 48 and 60 % sucrose. 5 ul of fraction 5 ml PE - mix well and pellet onto coverslips: 12,500 rpm for 15' at 4 deg.C in HB-4. Post fix in methanol for 5' and rehydrate and do 5051 antitubulin followed by anti-mouse, anti-human secondaries. Assess peak by concentration of double staining dots, pool and freeze in liq N2 in 10 ul aliquots.

    Preparation of Rat Liver Cytosol

    Equipment

    • 20 ml glass homogenizer equipped with Teflon pestle
    • Homogenizer or Drill press
    • Superspeed centrifuge with the equivalent of Sorvall SS-34 rotors
    • 50 ml polycarbonate centrifuge tubes
    • Ultracentrifuge with the equivalents of Beckman 50.2Ti and SW-28 rotors
    • 31.5 ml thick-walled polycarbonate ultracentrifuge tubes with screw caps
    • 25 ml Ultraclear ultracentrifuge tubes
    1. Chill the superspeed centrifuge, ultracentrifuge, SS-34 and 50.2Ti rotor to 4oC.
    2. In the cold room, rinse the liver well in PBS, then rinse in freshly prepared Homogenization buffer.
    3. Weigh the liver, return to the cold room, and finely mince the tissue with scissors. Transfer the minced tissue to a glass homogenizer, add 1 volume of homogenization buffer containing 0.5 mM MgGTP. Keeping the homogenizer immersed in slushy ice, homogenize with a Teflon pestle by 6 slow passes at 3,000 rpm.
    4. Transfer the homogenate to 31.5 ml centrifuge tubes, and remove cellular debris by centrifugation at 10,000 x g for 10 min at 4oC in a SS-34 rotor.
    5. In the cold room, collect the supernatant, transfer it to 31.5 ml ultracentrifuge tubes, pair them by weight, and clarify the cytosol by centrifugation at 100,000 x g for 60 min at 4oC in a 50.2Ti rotor.
    6. Collect the clarified cytosolic supernatant, disburse into 50 ul aliquots, and freeze by immersion in liquid nitrogen. Store at -80oC until use.
    • 20 ml glass homogenizer equipped with Teflon pestle
    • Homogenizer or Drill press
    • Superspeed centrifuge with the equivalent of Sorvall SS-34 rotors
    • 50 ml polycarbonate centrifuge tubes
    • Ultracentrifuge with the equivalents of Beckman 50.2Ti and SW-28 rotors
    • 31.5 ml thick-walled polycarbonate ultracentrifuge tubes with screw caps
    • 25 ml Ultraclear ultracentrifuge tubes

    Preparation of Rat Liver Cytosol

    Preparation of Rat Liver Organelle Fractions.

    Preparation of Rat Liver Cytosol

    1. Chill the superspeed centrifuge, ultracentrifuge, SS-34 and 50.2Ti rotor to 4oC.
    2. In the cold room, rinse the liver well in PBS, then rinse in freshly prepared Homogenization buffer.
    3. Weigh the liver, return to the cold room, and finely mince the tissue with scissors. Transfer the minced tissue to a glass homogenizer, add 1 volume of homogenization buffer containing 0.5 mM MgGTP. Keeping the homogenizer immersed in slushy ice, homogenize with a Teflon pestle by 6 slow passes at 3,000 rpm.
    4. Transfer the homogenate to 31.5 ml centrifuge tubes, and remove cellular debris by centrifugation at 10,000 x g for 10 min at 4oC in a SS-34 rotor.
    5. In the cold room, collect the supernatant, transfer it to 31.5 ml ultracentrifuge tubes, pair them by weight, and clarify the cytosol by centrifugation at 100,000 x g for 60 min at 4oC in a 50.2Ti rotor.
    6. Collect the clarified cytosolic supernatant, disburse into 50 ul aliquots, and freeze by immersion in liquid nitrogen. Store at -80oC until use.
    1. Perform steps 1-4 as in "Preparation of Rat Liver Cytosol" above except in step 2, homogenize the minced liver in homogenization buffer containing 0.5 mM MgGTP and 0.25 M sucrose.
    2. In the cold room, collect the supernatant and add 2.3 M sucrose stock to make the final concentration 1.25 M sucrose.
    3. Set up a sucrose density step gradient in a 25 ml ultraclear ultracentrifuge tube. All sucrose solutions should be made by dilution of the 2.3 M sucrose stock solution with homogenization buffer containing 0.5 mM MgGTP and chilled to 4oC. Add a 2 ml 2.0 M sucrose cushion to the bottom of the tube. Being careful not to disturb the cushion, layer 12 ml of clarified homogenate containing 1.25 M sucrose on the 2.0 M sucrose by using a pipette and allowing the solution to slowly run in a steady stream down the side of the centrifuge tube. Then add a 12 ml layer of 1.1 M sucrose, and finally an 8 ml layer of 0.25 M sucrose. Discard any excess homogenate. Pair the gradient by weight with a balance tube.
    4. Separate the organelles by density by centrifuging the gradient at 100,000 x g for 3 h at 4oC in a SW-28 rotor.
    5. In the cold room, with a Pasteur pipette carefully harvest the off-white band of Golgi membrane at the 0.25 M/1.1M sucrose interface and the off-white band of ER membrane at the 1.1 M/1.25 M sucrose interface and place them in separate 31.5 ml ultracentrifuge tubes on ice.
    6. Perform a Bradford assay to determine protein concentration of the Golgi and ER membrane fractions.
    7. Dilute both membrane fractions with 3 volumes of 0.25 M sucrose in homogenization buffer containing 0.5 mM GTP, and pellet the membranes by centrifugation at 100,000 x g for 1 h at 4oC in a 50.2Ti rotor.
    8. In the cold room, discard the supernatants and separately resuspend the dense, sticky ER and Golgi membrane pellets by extensive trituration in PM buffer containing 0.25 M sucrose and 0.5 mM GTP to achieve a protein concentration of 5 mg/ml.
    9. Disburse into 20 ul aliquots and freeze by immersion in liquid nitrogen. Store at -80oC until use.
    10. Determine the proper dilution of ER of Golgi membranes for use in the MT/Organelle motility assay. View various dilutions of membranes in simple perfusion chambers by VE-DIC microscopy. Note the dilution required so that ~20% of the area of the microscopic field is covered with organelles. For setting up the membrane MT motility assay, you will make need a stock that is 6 x the dilution just determined, which will be called 6X MEMBRANES.
    1. Chill the superspeed centrifuge, ultracentrifuge, SS-34 and 50.2Ti rotor to 4oC.
    2. In the cold room, rinse the liver well in PBS, then rinse in freshly prepared Homogenization buffer.
    3. Weigh the liver, return to the cold room, and finely mince the tissue with scissors. Transfer the minced tissue to a glass homogenizer, add 1 volume of homogenization buffer containing 0.5 mM MgGTP. Keeping the homogenizer immersed in slushy ice, homogenize with a Teflon pestle by 6 slow passes at 3,000 rpm.
    4. Transfer the homogenate to 31.5 ml centrifuge tubes, and remove cellular debris by centrifugation at 10,000 x g for 10 min at 4oC in a SS-34 rotor.
    5. In the cold room, collect the supernatant, transfer it to 31.5 ml ultracentrifuge tubes, pair them by weight, and clarify the cytosol by centrifugation at 100,000 x g for 60 min at 4oC in a 50.2Ti rotor.
    6. Collect the clarified cytosolic supernatant, disburse into 50 ul aliquots, and freeze by immersion in liquid nitrogen. Store at -80oC until use.

    Preparation of Rat Liver Organelle Fractions.

    Preparation of Rat Liver Organelle Fractions.

    1. Perform steps 1-4 as in "Preparation of Rat Liver Cytosol" above except in step 2, homogenize the minced liver in homogenization buffer containing 0.5 mM MgGTP and 0.25 M sucrose.
    2. In the cold room, collect the supernatant and add 2.3 M sucrose stock to make the final concentration 1.25 M sucrose.
    3. Set up a sucrose density step gradient in a 25 ml ultraclear ultracentrifuge tube. All sucrose solutions should be made by dilution of the 2.3 M sucrose stock solution with homogenization buffer containing 0.5 mM MgGTP and chilled to 4oC. Add a 2 ml 2.0 M sucrose cushion to the bottom of the tube. Being careful not to disturb the cushion, layer 12 ml of clarified homogenate containing 1.25 M sucrose on the 2.0 M sucrose by using a pipette and allowing the solution to slowly run in a steady stream down the side of the centrifuge tube. Then add a 12 ml layer of 1.1 M sucrose, and finally an 8 ml layer of 0.25 M sucrose. Discard any excess homogenate. Pair the gradient by weight with a balance tube.
    4. Separate the organelles by density by centrifuging the gradient at 100,000 x g for 3 h at 4oC in a SW-28 rotor.
    5. In the cold room, with a Pasteur pipette carefully harvest the off-white band of Golgi membrane at the 0.25 M/1.1M sucrose interface and the off-white band of ER membrane at the 1.1 M/1.25 M sucrose interface and place them in separate 31.5 ml ultracentrifuge tubes on ice.
    6. Perform a Bradford assay to determine protein concentration of the Golgi and ER membrane fractions.
    7. Dilute both membrane fractions with 3 volumes of 0.25 M sucrose in homogenization buffer containing 0.5 mM GTP, and pellet the membranes by centrifugation at 100,000 x g for 1 h at 4oC in a 50.2Ti rotor.
    8. In the cold room, discard the supernatants and separately resuspend the dense, sticky ER and Golgi membrane pellets by extensive trituration in PM buffer containing 0.25 M sucrose and 0.5 mM GTP to achieve a protein concentration of 5 mg/ml.
    9. Disburse into 20 ul aliquots and freeze by immersion in liquid nitrogen. Store at -80oC until use.
    10. Determine the proper dilution of ER of Golgi membranes for use in the MT/Organelle motility assay. View various dilutions of membranes in simple perfusion chambers by VE-DIC microscopy. Note the dilution required so that ~20% of the area of the microscopic field is covered with organelles. For setting up the membrane MT motility assay, you will make need a stock that is 6 x the dilution just determined, which will be called 6X MEMBRANES.
    1. Perform steps 1-4 as in "Preparation of Rat Liver Cytosol" above except in step 2, homogenize the minced liver in homogenization buffer containing 0.5 mM MgGTP and 0.25 M sucrose.
    2. In the cold room, collect the supernatant and add 2.3 M sucrose stock to make the final concentration 1.25 M sucrose.
    3. Set up a sucrose density step gradient in a 25 ml ultraclear ultracentrifuge tube. All sucrose solutions should be made by dilution of the 2.3 M sucrose stock solution with homogenization buffer containing 0.5 mM MgGTP and chilled to 4oC. Add a 2 ml 2.0 M sucrose cushion to the bottom of the tube. Being careful not to disturb the cushion, layer 12 ml of clarified homogenate containing 1.25 M sucrose on the 2.0 M sucrose by using a pipette and allowing the solution to slowly run in a steady stream down the side of the centrifuge tube. Then add a 12 ml layer of 1.1 M sucrose, and finally an 8 ml layer of 0.25 M sucrose. Discard any excess homogenate. Pair the gradient by weight with a balance tube.
    4. Separate the organelles by density by centrifuging the gradient at 100,000 x g for 3 h at 4oC in a SW-28 rotor.
    5. In the cold room, with a Pasteur pipette carefully harvest the off-white band of Golgi membrane at the 0.25 M/1.1M sucrose interface and the off-white band of ER membrane at the 1.1 M/1.25 M sucrose interface and place them in separate 31.5 ml ultracentrifuge tubes on ice.
    6. Perform a Bradford assay to determine protein concentration of the Golgi and ER membrane fractions.
    7. Dilute both membrane fractions with 3 volumes of 0.25 M sucrose in homogenization buffer containing 0.5 mM GTP, and pellet the membranes by centrifugation at 100,000 x g for 1 h at 4oC in a 50.2Ti rotor.
    8. In the cold room, discard the supernatants and separately resuspend the dense, sticky ER and Golgi membrane pellets by extensive trituration in PM buffer containing 0.25 M sucrose and 0.5 mM GTP to achieve a protein concentration of 5 mg/ml.
    9. Disburse into 20 ul aliquots and freeze by immersion in liquid nitrogen. Store at -80oC until use.
    10. Determine the proper dilution of ER of Golgi membranes for use in the MT/Organelle motility assay. View various dilutions of membranes in simple perfusion chambers by VE-DIC microscopy. Note the dilution required so that ~20% of the area of the microscopic field is covered with organelles. For setting up the membrane MT motility assay, you will make need a stock that is 6 x the dilution just determined, which will be called 6X MEMBRANES.

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