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Ta的微管蛋白聚合

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    核心提示:I. Solutions & SuppliesBRB80 : 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH

    核心提示:We "recycle" tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquo

    核心提示:Notes:The key variable in MT spindown experiments is ATP. Under high ATP conditions,conventional MAPs are selectively

    核心提示:I. Tubulin Prep OutlinePre-Prep: 1. Call slaughterhouse and request fresh brains to be picked up the morning of pre

    100 mM GTP

    We "recycle" tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day use. We generally store recycled tubulin in Injection Buffer without free GTP. We do this because depolymerization appears to be much better in IB, IB is ideal for microinjections/adding tubulin to extracts, and the absence of free GTP makes polymerization with GMPCPP, a very useful GTP analog that has ~5-10X lower affinity than GTP for tubulin, relatively straightforward.

    Notes:

    The key variable in MT spindown experiments is ATP. Under high ATP conditions,conventional MAPs are selectively co-sedimented with microtubules. In theabsence of ATP , both motors and MAPs will bind to MTs and pellet with themicrotubules. For MAP and motor analysis, I often supplement the extract with exogenous taxol-stabilized MTs to ensure that binding sites are not limitingand I am not seeing competition effects. For a unknown protein, it is best totry a variety of conditions. Below is a protocol for MAP pelleting under high ATP conditions. I have listed the modifications for MAP/motor protocolafterwards.

    I. Tubulin Prep Outline

    Pre-Prep:

    1. Call slaughterhouse and request fresh brains to be picked up the morning of prep

    2. Pour and equilibrate phosphocellulose column

    3. Make buffers

    4. Ensure that reagents, such as ATP and GTP, are present in sufficient

    amount for the prep

    1. Sign up for centrifuges and rotors and gather centrifuge tubes,

    blenders and motorized homogenizer/dounce

    1. Prepare cooler for transporting brains night before prep and organize

    coldroom for morning mayhem

    Prep Day:

    1. Remove meninges, brain stems and blood clots, weigh and

    homogenize brains in blenders

    1. Clarify homogenate and use supernatant for 1st polymerization cycle

    2. Collect 1st cycle polymer fraction by centrifugation

    3. Depolymerize 1st cycle polymer by homogenization at 0-40C

    4. Clarify depolymerization mix and use supernatant for 2nd

    polymerization cycle

    1. Collect 2nd cycle polymer fraction by centrifugation

    2. Depolymerize 2nd cycle polymer by homogenization at 0-40C

    3. Clarify depolymerization mix and load supernatant onto PC

    column

    1. Collect flowthrough from PC column, aliquot and freeze at -800C

    100 mM GMPCPP

    I. Solutions & Supplies

    5X BRB80: 10X IB: 500 mM K-glutamate; 5 mM MgCl2, Cushion: 60% glycerol in 1X BRB80 100 mM GTPGlycerolSmall Dounce 50.2Ti rotor

    TLA100.2 or 100.3 rotor II. Recycling Protocol1. Thaw 3-4 3 ml PC column fractions at 37¡C. Transfer to ice and mix in a 50 ml conical.2. Add BRB80 to 0.5X, 4 mM MgCl2 and 1 mM GTP. This is assuming that the tubulin aliquots are in CB . Thus, the final buffer composition is 90 mM K-Pipes, pH 6.7, 1.5 mM EGTA, 4.2 mM MgCl2,1 mM GTP. Mix by swirling and store on ice for 5'.3. Transfer to 37¡C. After 2' add half volume of glycerol . Mix the glycerol in by gentle vortexing. Incubate at 37¡C for 40'.4. Layer on a warm cushion in two 50.2Ti tubes. Spin for 45' at 40K at 35¡C in a 50.2 rotor.5. Aspirate sample, rinse sample cushion interface with warm IX IB 2-3 times. Aspirate completely and rinse the tubulin pellet 2X with warm 1X IB to remove any residual glycerol. Do this while the pellet is held in a 37¡C bath -- fill tube ~half-way with warm IB , then aspirate and repeat. This greatly reduces glycerol and GTP contamination in the final tubulin stock.6. Resuspend pellet in 1-2 ml of ice cold IB . Transfer the chunky pellet in IB to an ice-cold 2 ml dounce. Gently dounce on ice to break up the chunks. Incubate on ice for 30'. During this cold depolymerization, keep douncing gently every 2'-3'. Gentle sonication can also be used to break up the pellets.7. Spin 90K in TLA100.2 or TLA100.3 rotor at 2¡C for 10'-15' to clarify the depolymerization mix.8. Collect supernatant on ice and measure A280 of a 1/100 dilution in IB. Calculate concentration of tubulin using an extinction coefficient at 280 nm of 115,000 M-1cm-1. Freeze in 10-50 µl aliquots in liquid nitrogen and store at -80¡C.

    MAP pelleting:

    1. Prespin extract in TLA100.3 at 70K for 20' at 4 deg.C. Transfer supe to tube on ice.
    2. Extracts must have a source of GTP. For Xenopus egg extracts, we simply add an energy regeneration mix to extracts and the extract maintains physiologicalGTP levels. For dilute tissue culture extracts, it is best to supplement theextract with 0.5 mM MgGTP. This can be done before or after the prespin.
    3. Add 2 mM MgATP to extracts, warm to RT and add taxol to 5 uM. Mix well andincubate for 2'-3' before adding an additional 15 uM taxol . Incubate for 20'-30' at RT - 37 deg.C. .
    4. Layer the polymerized mixture onto a 1M sucrose cushion in BRB80 containing 0.5 mM ATP, 10 uM taxol and protease inhibitors and pellet in a TLS55 at 40K for 20' at 22 deg.C. You can also use a fixed angle rotor such as a TLA100.3 or TLA100.4. I like to pellet MTs at 100-150,000 g for 20 - 30' in TLA100 rotors over 30-40% glycerol/sucrose cushions. For larger ultra rotors, increase the speed and/or time to reach an equivalent clearing factor.
    5. Save supe for gel/blot, aspirate cushion while washing 2-3x with BRB80 and remove as much of the cushion as possible .
    6. Boil pellet in sample buffer and analyze.

    II. Buffers & Nucleotides

    PB : 0.1 M K-Pipes, pH 6.8, 0.5 mM MgCl2, 2 mM EGTA, 0.1 mM EDTA, 0.1 % b-mercaptoethanol, 1 mM ATP. Need 8 liters in coldroom CB : 50 mM K-Pipes, pH 6.8; 1 mM EGTA; 0.2 mM MgCl2. Need ~25 liters for equilibration, running and storage of PC column CB 1 M KCl: Need ~10 liters for prewashing and eluting the PC column To make 1L of 10X CB:151.2 grams PIPES, free acid3.8 grams EGTA2 ml of 1 M MgCl2pH with KOH to pH 6.75, and bring up to 1 liter.Check pH at 1X is 6.7 Make 3.5 liters of 10X CB for 10-12 brain prep. GTP: Sigma Type IIS- # G-8752ATP: Sigma Grade 1- # A-2383 Glycerol: 2-3L prewarmed to 37¡C

    Taxol: 10 mM stock; 100 µM, 10 µM and 1 µM dilutions all in anhydrous DMSO; Taxol is sold under the tradename "Paclitaxel" by Sigma

    MAP and motor pelleting:

    1. do not add extra ATP.
    2. add 2 -5 mM MgAMPPNP and/or supplement the extract with 15 U/ml hexokinaseand 20 mM glucose .
    3. supplement extract with taxol-stabilized MTs to 0.2-0.3 mg/ml finalconcentration after adding taxol to warmed up extract as above. Thisconcentration is for concentrated Xenopus extracts - could use 0.1 mg/ml fortissue culture cell extracts.

    III. Pouring a 1L Phosphocellulose Column

    Resin: Whatman P11 Cellulose Phosphate -- fibrous cation exchangerSummary: To pour a 1L column, start with 220 grams dry resin divided into 5 aliquots of 44 grams. Treat each aliquot with acid/base in a 2L beaker as described below. Older procedures described the use of large Buchner funnels to rapidly remove the acid/base. However, gentle stirring of the resin with a plastic/glass rod to suspend it in a 2L beaker, followed by settling of the resin for 5' by gravity has worked well for us. This method also incorporates de-fining of the resin into the acid/base cycling protocol. After acid/base treatment the resin is washed well, packed, treated with BSA to block irreversible binding sites and equilibrated for use.Solutions & Supplies:220 grams Phosphocellulose5L 0.5N NaOH5L 0.5N HCl13L 0.5M K-Phosphate, pH 6.85L ddH205 2L beakers12L CB 1M KCl20L CB300 ml of 30 mg/ml BSA in CB 1-1.5L cleaned column housing2 stirring rods2 aspirators with large trapsPeristaltic pump10 ml plastic pipets as inlets for peristaltic pump

    Column Preparation Procedure:1. Pour 1L 0.5N NaOH into 5 x 2L beakers. Add 44 grams PC to each beaker stirring gently with a rod until the PC is wetted and an even slurry is present. Let stand at room temperature for 5'.2. Aspirate off supernatant, including fines, and quickly add 1L 0.5M K-phosphate to neutralize, gently mixing with a rod. Check that pH is ~7 and let stand 5'.3. Aspirate off supernatant, add 1L ddH20 and gently stir to resuspend settled resin.4. Allow the resin to settle.5. Aspirate off supernatant, add 1L 0.5N HCl, gently stir to resuspend and wait 5'.6. Aspirate off supernatant, add 1L 0.5M K-phosphate, stir and check pH is 7.7. After resin has settled, aspirate supernatant and combine all the resin in a 4L beaker. Use the remaining 0.5M K-phosphate to wash the resin by resuspending, letting settle and aspirating the supernatant.8. Wash 3 x 1L CB 1M KCl as done in 7.9. In the cold room, pour the resuspended resin into the column housing and pack by pumping from the bottom . Pack at 45 ml/hour/cm cross-sectional area. For a 5 cm diameter column this is ~880 ml/hour or ~14.5 ml/min. After resin is packed, switch to pumping from the top. Run 7L of CB 1M KCl through the column at 5-10 ml/min.10. Wash with 10L CB. Check conductivity to ensure that all the KCl is gone. The resin may expand as the salt is washed out so make sure there is a large buffer head on the resin bed.11. Load 300 ml of 30 mg/ml BSA in CB, follow with 700 ml CB and stop the column. Leave the column sitting for 2 hr during which the BSA blocks irreversible binding sites on the resin -- this is very important the first time a column is used to prevent loss of the tubulin.12. Wash the column with 2L CB 1M KCl to elute BSA that is not irreversibly bound.13. Wash column with 10L of CB. The column is now ready for use.

    II. Prepolymerization Clarification

    Although not absolutely necessary, we recommend mixing tubulin and nucleotides in 1X BRB80 for 5' on ice and then clarifying this mix using a TLA100 rotor for 5' at 90K at 2¡C. We especially recommend this clarification when polymerization includes GMPCPP and/or highly labeled fluorescent tubulins. We also recommend a clarification spin on thawed labeled tubulins prior to microinjection.

    Note on Buffers:

    I have done this with extracts prepared using BRB80 and XB and find it works with both. I think it is best to try YourFavorite Buffer and BRB80 side-by-side to maximize chances ofsuccess. I ended up using XB since I was trying to relate MAP profiles to cell cycle-dependent MT dynamics in XB extracts. The buffer in the cushion is notvery important.

    Note on Taxol stabilized MTs:

    1. Dilute tubulin to 2 mg/ml tubulin in BRB80 1 mM GTP 1 mM DTT on ice; incubate 5'.
    2. Warm to 37 deg.C, add 1/100 vol of 0.02 mM taxol in DMSO; 5' at 37 deg.C
    3. Add 1/100 vol of 0.2 mM taxol; 5' at 37 deg.C
    4. Add 1/100 vol of 2 mM taxol; 10-15'' at 37 deg.C. These MTs can be stored at RT for upto 1 week.

    IV. Brains

    It is essential to get fresh brains -- yields decline significantly if the brains have been stored for a while after removal. Frozen brains do not work for preparing tubulin. The best preps have been done with freshly removed brains transported in an ice-filled cooler to the lab within 1-2 hours of removal. For transporting 10-12 brains from the slaughterhouse, we use 2 size 16 Coleman coolers containing 3 liters of cooled 1.5% NaCl to which one large bag of party ice is added on the way to the slaughterhouse.

    III. GTP Polymerization

    1. On ice mix unlabeled tubulin and labeled tubulin at an appropriate ratio in 1X BRB80 with 1 mM DTT and 1 mM GTP. Incubate at 0¡C for 5'.

    2. Clarify mix in TLA100 rotor at 90K for 5' at 2¡C.

    3. Collect supernatant and incubate at 37¡C. If the tubulin concentration is 2 mg/ml or higher, assembly will proceed rapidly to steady state . If the concentration is lower, nucleation can be limiting and the precise kinetics of approach to steady state is difficult to predict and will depend on the amount of active tubulin in your mix. For many experiments, we add seeds to surmount the nucleation barrier, thereby specifically assaying elongation -- for this we routinely make GMPCPP seeds, wash out any free GMPCPP and add a small volume of the seeds after the polymerization mix has been at 37¡C for 1'. Axonemes or centrosomes can also be used as nucleating structures. If the purpose is labeling or recycling the tubulin, polymerization is promoted by addition of DMSO or glycerol as described above.

    V. Centrifuges & Rotors

    6 Sorvall RC-5C or equivalent lowspeed centrifuges

    6 GSA or equivalent rotors

    4 Beckman ultracentrifuges

    4 Type 19 rotors

    2 Type 35 rotors

    2 Type 45Ti rotors

    1 Type 50.2Ti rotor

    cold = 4¡C

    warm = 37¡C

    IV. Taxol Polymerization

    1. On ice mix unlabeled tubulin and labeled tubulin at an appropriate ratio in 1X BRB80 with 1 mM DTT and 1 mM GTP. Incubate at 0¡C for 5'.

    2. Clarify mix in TLA100 rotor at 90K for 5' at 2¡C. Incubate supernatant at 37¡C for 1'-2'.

    Now there are two options :

    I. Add taxol stepwise to equimolar as follows :

    Add 1/10 vol 1 µM taxol; Incubate at 37¡C for 5'-10'

    Add 1/10 vol 10 µM taxol; Incubate at 37¡C for 5'-10'

    澳门金莎娱乐网址,Add 1/10 vol 100 µM taxol; Incubate at 37¡C for 15'

    Pellet microtubules over a warm 40% glycerol in BRB80 cushion in a TLA100, 100.2 or 100.3 rotor, aspirate and wash sample/cushion interface, rinse pellet and resuspend in warm BRB80 1 mM DTT 10-20 µM taxol

    Note: If taxol is added all at once it will cause tubulin precipitation! If polymerizing 2 mg/ml tubulin, use 2 µM, 20 µM and 200 µM steps

    OR II. Add 10% DMSO and incubate at 37¡C for 20'-30'

    Pellet and resuspend microtubules as described above

    For DMSO polymerization it is best to have high tubulin concentrations in the original mix before adding DMSO. However, MTs must be resuspended after pelleting with equimolar taxol.

    A "quick-and-dirty" taxol polymerization method :

    1. Thaw recycled tubulin stored in IB

    2. Add equal volume 2X BRB80 2 mM DTT 2 mM GTP 20% DMSO

    3. Incubate at 37¡C for 20'-30'

    4. Pellet microtubules and resuspend as described above

    If there is labeled tubulin in the mix, dilute the microtubules to 1-10 µg/ml and check under a fluorescent microscope. Taxol-stabilized microtubules can be sheared by diluting them to ~100 µg/ml and then passing them through a 27g needle ~5-6 times. All dilutions of taxol-stabilized microtubule should be done into buffers containing 10-20 µM taxol.

    VI. Protocol

    1. In the cold room, remove meninges , blood clots, and brain stems; weigh the brains and homogenize with equal volume of PB for 3 x 15s in a Waring blender.2. Collect homogenate , transfer into 36 GSA bottles and spin 90' at 12K in a GSA rotor at 4¡C.3. Collect supernatant and transfer 1 liter to a 1.8L glass Fernbach flask that has 500 ml of 37¡C glycerol. Add 0.1 mM GTP, 0.5 mM ATP, and 3.5 mM MgCl2 . The ATP and GTP are added as solids. Hold the flask in a warm water-filled sink and swirl constantly to dissolve the solids and to mix in the glycerol. Transfer to a 37¡C bath, monitor temperature of mixture using a clean thermometer and polymerize for 60' after the temperature of the sample has reached 32¡C. The approach to 37¡C can be accelerated by swirling the flask in a large hot water reservoir -- constant swirling is essential in this case to disperse the heat evenly and care must be taken to avoid overheating the mixture.4. Transfer the polymerization mixture to Type 19 bottles, and spin for 2.5 hrs at 19K in 4 Type 19 rotors at 35¡C. Use an additional Type 35 at 17.5K for 2.5 hrs if necessary. At the end of the spin set centrifuges to 4¡C.5. Decant and discard supernatant. In the cold room, resuspend the gelatinous pellets in PB aiming for a final volume of ~700-800 ml. We use ~40-50 ml for 3 tubes, sequentially removing the pellets from each tube using a plastic scraper and making sure that all tubes get rinsed once or twice after the majority of the large gelatinous pellet has been removed. To homogenize the chunky pellet resuspension, we use a Yamato "pour-through" continuous flow homogenizer -- this is a device that drives a motorized teflon pestle in a funnel shaped glass barrel. Mixtures poured on top get homogenized by the pestle as they travel through the middle of the barrel and come out the bottom. We Yamato the chunky pellets till the resuspension is a smooth yellow liquid of ~700-800 ml total volume. After all the pellets are homogenized, we depolymerize on ice for ~30' during which we continue Yamatoing the mixture once every 3'-5'. A large motorized teflon dounce or large tip sonicator can be used as alternatives to the Yamato for resuspending the chunky pellets. Check protein concentration by Bradford using BSA as a standard. If >20 mg/ml , dilute to 20 mg/ml.6. Spin the depolymerization mixture 30' at 35K in 2 Type 45 rotors at 4¡C. At the end of the spin set the centrifuges to 35¡C.7. Decant supernatant into a 1L graduated cylinder in coldroom and measure volume. Pour into a 1.8L Fernbach flask, add half volume of 37¡C glycerol, solid GTP to 0.5 mM final and MgCl2 to 4 mM final . Set up polymerization as described in 3. above. Polymerize for 40' after temperature of mixture has reached 32¡C.8. Spin the polymerization mixture at 35 K at 35¡C for 1 hr in 2 Type 35s 1 Type 45. Make sure one chilled centrifuge is available for the next spin.9. Discard supernatant and resuspend pellets in a final volume of ~100-150 ml of CB, as described above in 5. Protein concentration by Bradford should not be more than 25 mg/ml. Depolymerize on ice for 40' and then spin the depolymerization mix 30' at 40K in a 50.2Ti rotor at 4¡C.10. Collect supernatant, measure concentration by Bradford and load onto the PC column . After the sample is loaded and ~150 ml buffer has flowed through start collecting fractions. The eluted tubulin will be apparent by its slight yellowish tinge. Measure concentration by Bradford using BSA as a standard and pool such that the final concentration is between 5-10 mg/ml. Mix pool on ice, make 3 ml aliquots in 5 ml snapcap polypropylene tubes and freeze in liquid nitrogen. Store frozen aliquots at -80¡C. The entire procedure, from time of arrival of brains till freezing of the tubulin will take ~17-18 hours. The next day, run 3 volumes of CB 1M KCl to elute MAPs from the PC column , and then equilibrate column into CB 0.1% azide for storage. Phosphocellulose will lose capacity when stored wet -- this can be reduced by storage in a phosphate buffer containing 0.1% azide.

    V. GMPCPP Polymerization

    GMPCPP is the best current GTP analog for tubulin polymerization. Its major limitation is lack of commercial availability. In the presence of potassium as counterion, GMPCPP is very slowly hydrolyzed within the microtubule lattice, and is essentially non-hydrolyzable within the time course of most experiments. In the presence of sodium as counterion, GMPCPP is hydrolyzed slightly faster in the lattice -- this hydrolysis is accelerated tremendously by treatment with glycerol. Given this information on the effect of buffer counterions on GMPCPP stability within microtubule lattices, we always use potassium counterion buffers for all our microtubule work. GMPCPP is a potent nucleator of microtubules. Therefore, at tubulin concentrations of 1 mg/ml or higher, very numerous and short microtubules are formed in the presence of GMPCPP. If longer GMPCPP microtubules are desired, nucleation can be limited by diluting the tubulin to ~2-3 µM . We generally make a 1-3 mg/ml CPP tubulin mix and store it at -80¡C in small aliquots. Directly polymerizing this mix results in short GMPCPP seeds. Diluting the mix while thawing it results in formation of longer CPP microtubules.

    1. On ice mix unlabeled tubulin and labeled tubulin at an appropriate ratio in 1X BRB80 with 1 mM DTT and 0.5-1 mM GMPCPP. Incubate at 0¡C for 5'-10'.

    2. Clarify mix in TLA100 rotor at 90K for 5' at 2¡C. Freeze supernatant in 5-10 µl aliquots in liquid nitrogen and store at -80¡C.

    3A. To form short GMPCPP seeds, transfer a tube from the freezer to a 37¡C bath. Incubate 15'-20' at 37¡C. Dilute to 150-200 µl with warm BRB80 1 mM DTT, pellet the seeds in a TLA100 rotor , discard supernatant and resuspend pellet in 1-2X the starting volume of BRB80 1 mM DTT. This process removes free CPP and any unpolymerized tubulin. Seeds are generally added at 1/20-1/50 vol to a polymerization mix containing tubulin and 1 mM GTP. Given the ~10X higher affinity of tubulin for GTP versus GMPCPP, the amount of GMPCPP added from a seed mix at these dilutions is insignificant. Therefore, we often add seeds directly into a polymerization mix without dilution/sedimentation/resuspension.

    3B. To form long GMPCPP microtubules, thaw a CPP mix tube by adding in enough warm BRB80 1 mM DTT such that the final tubulin concentration is 2-3 µM . Incubate at 37¡C for 30' or longer. Free CPP can be removed as described in 3A or the CPP microtubules can be used directly for assays.

    VI. Determining Concentration of GMPCPP/Taxol MTs

    To accurately estimate the concentration of tubulin polymer in GMPCPP/Taxol polymerizations, MTs are pelleted and resuspended in buffer without free nucleotide. A small amount of the resuspended MTs are then diluted into a buffer containing CaCl2 on ice to induce depolymerization and tubulin concentration determined by the A280. Here is a protocol for GMPCPP MTs :1. Pellet polymerization mixture 90K 5' in TLA100 at 35¡C.2. Remove supe as thoroughly as possible.3. Resuspend pellet in 80% of starting volume using 37¡C BRB80 1 mM DTT until homogenous 4. Dilute 10 µl resuspended MTs into 90 µl of BRB80 50 mM KCl 5 mM CaCl2 and incubate on ice for 10' .5. After 10' at 0¡C, read A280 against blank and calculate concentration using an extinction coefficient of 115,000 M-1cm-1.

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