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澳门金沙app肌球蛋白轻链的制备,大范围微管蛋

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

    骨干提醒:Large Scale Tubulin Preparation Tubulin is purified from bovine/porcine brain by two cycles of polymerization/depolymer

    宗旨提醒:Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highes

    主干提醒:To be done the day before:1. Put meat grinder and accessories in the cold room.2. Put 6 one-liter centrifuge bottles inTo be done the day before:1. Put meat grinder and accessories in the cold room.2. Put 6 one-liter centrifuge bottles in the cold room.3. Put 14 to 16 500 ml Beckman polycarb centrifuge bottles with lids and inserts in thecold room.4. Clean the 8 liter and the 28 liter beakers.5. Put ETOH in the cold room.6. Refrigerate or equilibrate the DE52 to be used.7. Find parts to and assemble the 5½ cm ID column.8. Make sure there is plenty of cold water in the cold room.9. Thaw 8 M urea.SolutionsBuffer A. Homogenizing Buffer: 28 liters: 50 mM KCl, 20 mM Tris , 15 mM MCE,0.2 mM MgAc2 Buffer B. Triton X-100 Buffer: 4 liters of Buffer A plus 40 ml X-100 Buffer C. Urea Homogenizing Buffer:2.4 liters: 8 M urea, 1 mM EDTA, 15 mM MCE, 20 mMTris Buffer D. ETOH/MCE: 3 liters: For skeletal Muscle - 95% ETOH, 15 mM MCE For Cardiac Muscle - Buffer E. Column Buffer: 16 liters:20 mM Tris, 15 mM MCEBuffer F. Elution Buffer: 4 liters: 0.150 M KCl, 10 mM Tris, 15 mM MCEBuffer G. Dialyzing Solution: 8 liters:4 mM MOPS , 15 mM MCEMCE = 2-mercaptoethanol .Day 11. Is cleaned of all fat and connective tissue, then sliced into strips and run through themeat grinder. Approximately 750 grams of tissue is needed.2. Tissue is weighed .3. The tissue is homogenized in 6 volumes of Buffer A using a large blender and blendingfor a total time of 90 seconds. It is best to bland in 30 second intervals with 30 secondrests until total blend time is 90 seconds.4. The homogenate is spun at 4200 rpm for 15 minutes in the Beckman J-6 using 1 literbottles.5. Discard the supernatant and place the pellets on ice. Add homogenizing Buffer A untilthe bottles are ½ full. Using the J-K homogenizer, resuspend the pellets 30 seconds.Fill the bottles with Buffer A and centrifuge at 4200 rpm for 15 minutes in the BeckmanJ-6.6. Repeat step 5 with Buffer B .7. Repeat step 5 four more times with Buffer A until the pellet is nearly white and thesupernatant is nearly clear .Discard supernatant and any loose white solid material which may be found on thecompacted white pellets.Day 28. Remove about 1 liter of pellets and place in the blender. Using about 500 ml of BufferC, rinse the bottles and add to the blender. Add 2 liters of Buffer C and approximately50 g solid urea and homogenize for 2 minutes in 30 second intervals in a largeblender.If frozen pellets are used, thaw the pellets in the cold room over-night. Put the pellet inthe blender the next morning and add 2 liters of Buffer C to blender and homogenizefor 2 minutes.9. Pour mixture into an 8 liter beaker. Rinse the blender with remainingBuffer C and add to beaker. Stir at room temperature for 2 hours using large bladeand drill .10. Add equal volume of Buffer D in the cold room and stir for 1 hour at 4°C.11. Centrifuge at 8000 rpm for 20 minutes at 4°C using the Beckman J21-C and 500 mlprechilled polycarb bottles with inner seals *usually 14 bottles

    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

    Large Scale Tubulin Preparation Tubulin is purified from bovine/porcine brain by two cycles of polymerization/depolymerization followed by removal of copurifying proteins on a phosphocellulose column. The procedure described here is for a large scale prep that yields 1-4 grams of tubulin. The protocol can be scaled down if such a large prep is either not necessary or not doable. For ease of organization, all the pre-prep and day-of-prep activities are listed in outline form before details about the prep itself.

    Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highest yielding preparation I have done. The protocol calls for 3 pig brains, and should yield ~ 60 mg of purified tubulin. Solutions and reagents: 100g P-11 cellulose phosphate fibrous cation exchanger 6L 0.1M HCl 6L 0.1 M NaOH 2L 0.1 M MgSO4 2L 10x Column Buffer ~10 L 1x column buffer 10 M NaOH 3 fresh pig brains 300 ml Homogenization buffer 1L PM buffer 100 mM MgATP 1L PMG Buffer 100 mM MgGTP 1M dithiothreitol Glutamic acid, sodium salt Equipment: Waring Blender Temperature controlled ultracentrifuge with the equivalent of a Beckman 50.2Ti rotor At least 24 31.5 ml thick-walled polycarbonate ultracentrifuge tubes with screw caps 37oC water bath 30 ml Dounce "A" glass homogenizer 2L scintered glass filter funnel and 2L sidearm Erlenmeyer flask Amicon 44 mm x250 mm #95240 or equivalent adjustable volume column for low pressure liquid chromatography Peristaltic pump Fraction collector UV monitor or Bradford reagent DAY 1: Preparation of the phosphocellulose column.

    • 3 spins are necessary;rotor JA10).12. Filter the supernatant through glass wool into an 8 liter beaker saving supernatant.Discard pellets and rinse bottles immediately with water. Soak bottles in the Beckmansoap.13. Add 800 ml of regenerated DE52 to the filtered supernatant. Let this absorb overnightwith stirring, at 4°C.Day 314. Stop stirring and allow the DE52 to settle . When a distinct line isvisible between the DE%2 and the liquid, siphon off as much ETOH supernatant aspossible.15. Divide the DE52 into 1 liter bottles, fill with Column Buffer E and centrifuge in the J-6for 10 minutes at 1000 rpm.16. Discard supernatant and refill bottles with Column Buffer E. Shake slightly so that theDE52 is resuspended in the buffer. Spin in the J-6 for 10 minutes at 1000 rpm.Repeat for a total of 4 washes.17. Pack the DE52 into the 5½ cm ID column in the cold room.18. When all of the DE52 has been put in the column and has packed somewhat, take a10 ? sample of the eluting fluid to see if any protein is still being eluted . When it has been determined that no more proteinis being eluted and the DE52 is packed, take the Buffer E off of the top of the columnwith a syringe. Then carefully layer the Elution Buffer F on top of the column to a heightof about 2 inches.19. Fill a 4 liter beaker with Elution Buffer F. Collect 20 ml fractions .The adaptor top to the column may need to be used if the DE52 is packed too low inthe column. This step will run overnight. Set the speed at about 1 drop/sec.Day 420. A. Run Coomassie Blue Assay on every other tube. Take 10 ? samples, add dye,and read against a water blank at A595 on the spectrophotometer; graph A595against fraction number. Absorbance should rise quickly, fall slightly, and thentail-off slowly. Pool tube under first sharp high protein peak and poolremaining tubes under broad lower protein peak . Find volumes ofPools 1 and 2.B. Measure conductivity of every fifth tube. Allow the conductivity meter to sit in thecold room for 30 minutes before using it n the cold room; graph mMHO againstthe fraction number.21. Put samples in lyophilizer bottles and freeze in liquid nitrogen. Put on lyophilizer andleave until dry .Day 522. Resuspend separate light chain pools 1 and 2 in a small volume of Dialyzing SolutionG. Pipette into dialyzing tubing through a small funnel. Tie three knots ineach end and put in 8 liters of DialyzingSolution G. Change to a fresh solution of Dialyzing Solution G after a few hours toforce a new equilibrium to occur. Dialyze overnight.Day 6 and after23. Centrifuge in J-21C using JA20 rotor at 15,000 rpm for 20 minutes. Take a smallsample for gel and assays and freeze the remainder in -60°C freezer .24. Determine the protein concentrations of Pools 1 and 2 with the Coomassie Blue Assayand B.S.A. standards. Run 10% SDS gels.25. Do 32P assay to determine maximal extent of phosphorylation of light chain.26. Scan gels with spectrophotometer at A 600. Cut out and weigh the peaks to determine% impurities and percent of light chain 1 and light chain 2 . Take aphotograph of the gels.

    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

    I. Tubulin Prep Outline

    1. Weigh out 90 g of Whatman P-11 phosphocellulose and add it to 2 L of 0.1 M NaOH in a 4 L beaker while mixing VERY gently with a glass rod. Mix the suspension gently with the rod for 5 minutes, then allow the solid to settle for 20 min. Aspirate off excess solution, and transfer the remaining phosphocellulose slurry to a 2L scintered glass funnel on a 2L sidearm Erlenmeyer flask that is connected to a vacuum line. Carefully vacuum filter the remaining 0.1 M NaOH from the phosphocellulose, BUT NEVER ALLOW THE RESIN TO RUN DRY!
    2. Gently scrape the phosphocellulose out of the funnel, and return it to the 4 L beaker, add 2 L of 0.1 M NaOH, mix gently for 5 min, and check the pH with pH indicator paper. Repeat the mixing settling, aspiration, and filtering of the resin as in step 1. If the pH of the slurry was not above 12, transfer the phosphocellulose to the 4 L beaker, add 2 L of 0.1M NaOH, and repeat the mixing, settling, aspiration, and filtering treatment as in step 1, leaving the wet phosphocellulose in the funnel.
    3. Rinse 4 L of distilled water through the phosphocellulose by vacuum filtration, again, never letting the resin run dry.
    4. 金沙国际娱乐网址,Transfer the phosphocellulose from the funnel to the 4 L beaker and add 2 L 0.1 M HCl. Mix gently, allow the resin to settle, aspirate off excess solution, and vacuum filter the resin as in step 1. Repeat the 0.1 M HCl treatment cycle until the pH of the phosphocellulose slurry is below 3.
    5. Rinse the phosphocellulose with 4 L of distilled water by vacuum filtration.
    6. Transfer the phosphocellulose resin to the 4 L beaker and add 2 L of 0.1 M MgSO4. Mix, settle, aspirate, and vacuum filter as in step 1.
    7. Transfer the phosphocellulose resin to the 4 L beaker, add 2 L of 10 x column buffer. Mix gently for 10-15 min, allow the resin to settle, aspirate excess column buffer off, and vacuum filter.
    8. Transfer the resin to the 4 L beaker, add 2 L of 1x column buffer, mix gently for 5 min, check the pH of the slurry with a pH meter, and adjust it to 6.6 with 10 M NaOH. Allow the resin to settle, aspirate excess buffer, and vacuum filter it. Repeat the 1x column buffer treatment cycle, adjusting the pH of the slurry each time to 6.6 with 10M NaOH, until the pH of the slurry is 6.6 without adjustment after resuspension and mixing. Allow the resin to settle for 20 min, aspirate off buffer until the settled resin:buffer ratio is 3:1 .
    9. Pour the column. Mix the resin gently until it is evenly suspended in the buffer, then rapidly pour the slurry into an empty 44 mm x 250 mm liquid chromatography column to fill it to the top. Cover the column with parafilm, and allow the resin to settle for several hours.
    10. Fill the column adjuster plunger with column buffer, and VERY slowly insert it into the column, being very careful not to disturb the phosphocellulose resin. As the adjuster plunger is inserted into the column, the column inlet tubing that is attached to the plunger should be immersed in a 4L reservoir of column buffer, and should expel all air bubbles as the adjuster plunger is inserted. Tighten and seal the adjuster plunger fittings, leaving the inlet tube in the buffer reservoir.
    11. Attach the outlet tubing to the peristaltic pump, and set the pump to run at 0.25 ml/min to allow the column to pack properly for the next ~48hrs while MT protein is prepared. A well-packed phosphocellulose column should be perfectly even in color with no evidence of "cracks" in the resin. The better the phosphocellulose column is packed, the more concentrated the peak of elution of tubulin protein.

    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.

    Pre-Prep:

    DAY 2: Cycling preparation of MT protein.

    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.

    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

    1. Keep the brains in an evacuated plastic ziplock bag buried in ice from the time of slaughter during transport back to the laboratory.
    2. In a 4oC cold room, carefully and thoroughly remove the meninges and any blood-red tissue from the surface, stem, and within the folds of each brain. Do this by both picking tissue away by hand and wiping with kimwipes. The dry wipe will stick to the meninges, peeling away the deep red membrane from the pinkish grey nervous tissue underneath as the wipe is gently drawn across the surface of the brain.
    3. Cut the cleaned brains into 2-3 cm2 cubes and weigh the tissue. Transfer the tissue to a Waring blender and add 0.5 ml/g freshly prepared Homogenization Buffer containing 1mM MgATP.
    4. Homogenize the tissue by blending 5 s on high speed and then 45 s on low speed. This should result in a suspension with the color and consistency of a strawberry milkshake.
    5. Using a 50 ml serological pipette with 1/3 of the tip cut off and a pipette bulb with strong suction, transfer the brain homogenate to several 31.5 ml polycarbonate ultracentrifuge tubes, note the homogenate volume, and pair the tubes by weight . Discard any extra homogenate that does not fit into a full rotor of centrifuge tubes. Tubulin is sensitive to proteases and easily denatured, and keeping extra homogenate for one hour during the following centrifugation step does not significantly increase the final yield.
    6. Centrifuge the homogenate at 100,000 x g for 60 min at 4oC in a 50.2Ti rotor to remove undisrupted tissue from cell cytosol.
    7. At room temperature, carefully collect the cytosolic supernatants from the tubes with a pipette, pool them in a graduated cylinder, dilute the total 1:1 with PMG, and add MgGTP to 0.2 mM to promote MT polymerization. Disburse into 31.5 ml ultracentrifuge tubes and pair them by weight.
    8. Immerse the portion of the tubes containing the cytosol in a 37oC water bath and allow MT protein to polymerize into MTs by incubating for 45 minutes. During this polymerization incubation, warm the ultracentrifuge and 50.2Ti rotor to 25oC.
    9. Pellet MTs from the cytosol by centrifugation at 100,000 x g for 45 min at 25oC in a 50.2Ti rotor.
    10. In the cold room, discard the supernatant and resuspend the MT pellet in 1/5 the volume of homogenate in PM buffer containing 0.2 mM GTP. To do this, put a few mls of resuspension buffer into each centrifuge tube, as well as into a small glass Dounce homogenizer . Scrape out the sticky pellets with a round-ended weighing spatula and transfer them into the buffer in the homogenizer. Resuspend any pellet remaining in the ultracentrifuge tubes by trituration, and transfer it to the homogenizer. Homogenize the pellets by 5-10 passes with a type "A" pestle. Disburse the resuspended MTs into 31.5 ml ultracentrifuge tubes and pair them by weight.
    11. Incubate the resuspended MTs on ice for 30 minutes with gentle mixing every 5 min to allow for MT depolymerization. During this time chill the ultracentrifuge and 50.2Ti rotor to 4oC.
    12. Clarify the MT protein by centrifugation at 100,000 x g rpm for 45 min at 4oC in a 50.2Ti rotor.
    13. At room temperature, collect the supernatant containing the MT protein, dilute it 1:1 with PMG, add GTP to 0.2 mM, disburse it into 31.5 ml centrifuge tubes, and pair them by weight.
    14. Immerse the portion of the tubes containing the MT protein solution in a 37oC water bath and allow MTs to polymerize by incubation for 45 min. During the incubation, warm the ultracenrtifuge and 50.2Ti rotor to 25oC.
    15. Pellet the MTs from polymerization-incompetent tubulin by centrifugation at 100,000 x g for 45 min at 25oC in a 50.2Ti rotor.
    16. In the cold room, discard the supernatant. Add 1ml of column buffer containing 0.5 mM GTP to each tube, and being careful that the buffer is covering the pellet, immerse the ultracentrifuge tubes into liquid nitrogen to freeze the pellets. Store the tubes at -80oC until the next day or whenever phosphocellulose column purification of tubulin is to be carried out.

    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

    amount for the prep

    澳门金沙app,DAY 3: Phosphocellulose column purification of tubulin from MT protein

    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.
    1. Sign up for centrifuges and rotors and gather centrifuge tubes,
    1. Chill ultracentrifuge and 50.2Ti rotor to 4oC.
    2. Prepare and equilibrate the phosphocellulose column with column buffer containing 0.5 mM GTP and 1mM DTT. Turn off the peristaltic pump, and if the resin bed has settled, carefully loosen the seals on the adjuster plunger, slowly insert the adjuster plunger further until it barely touches the top of the resin, and retighten the seals. Switch the inlet tube from the reservoir of 1x column buffer to a 1L reservoir of 1x column buffer containing 0.5 mM MgGTP and 1 mM DTT, being careful not to introduce any bubbles into the inlet tube. Turn the peristaltic pump back on and adjust the speed to 1.8 ml/min. Allow at least 300 ml of buffer to be drawn through the column before loading the MT protein. During the phosphocellulose column equilibration, perform steps 3 and 4 below.
    3. Thaw the MT pellets by immersing the ultracentrifuge tubes in a 37oC water bath until the pellets turn from chalky white to completely translucent white. As soon as the pellets are thawed, place the tubes on ice immediately and take them into the cold room.
    4. Resuspend the pellets on ice with a Dounce glass homogenizer and type "A" pestle in ~3x their volume of 1x column buffer containing 0.2 mM GTP. Transfer the resuspended MTs to a 31.5 ml ultracentrifuge tube. Allow the MTs to depolymerize by incubation with gentle mixing every 5 min on ice for 30 min.
    5. Clarify the MT protein by centrifugation at 100,000 x g for 45 min at 4oC in a 50.2Ti rotor.
    6. In the cold room, collect the clarified MT protein supernatant and add MgGTP to a final concentration of 0.5 mM and DTT to 1mM.
    7. After column equilibration, switch the column inlet tube from the buffer reservoir to the clarified MT protein. Load the MT protein onto the column at 1.8 ml/min. When all of the MT protein is loaded, switch the inlet back to the buffer reservoir and begin to collect 10 ml fractions. Tubulin will pass through the column and come off after ~100 mls, while MT binding proteins will remain bound to the phosphocellulose resin.
    8. Monitor the elution of tubulin either with the UV monitor by absorption at 280 nm or by adding 100 ul of each aliquot to 1 ml of freshly prepared Bradford reagent and looking for blue color. As each aliquot comes off, add MgGTP to a final concentration of 1 mM . Pool fractions containing tubulin .
    9. At room temperature, add 0.186 g/ml glutamic acid to the tubulin solution and stir slowly until it is dissolved. Disburse the solution into 31.5 ml ultracentrifuge tubes and pair them by weight.
    10. Immerse the centrifuge tubes to the level of the liquid within in a 37oC water bath and incubate for 30 min to allow MTs to polymerize. During this time, warm the 50.2Ti rotor and ultracentrifuge to 25oC.
    11. Isolate the MTs from polymerization-incompetent tubulin by centrifugation at 100,000 x g for 30 min at 25oC in a 50.2Ti rotor.
    12. In the cold room, resuspend the MT pellets on ice with a Dounce glass homogenizer as above, in 3x their volume of PM buffer containing 0.5 mM MgGTP. Incubate the resuspended MTs on ice for 30 min to allow MT depolymerization. During this time, determine the tubulin concentration by measuring the absorbance at 280 nm using the extinction coefficient of tubulin = 115,000 Mol/cm /extinction coefficient)). Add PM buffer containing 0.5 mM MgGTP to adjust the final protein concentration to 45 uM.
    13. Disburse into several 1 ml aliquots and several 50ul aliquots , freeze by immersion in liquid nitrogen, and store at -80oC until use.

    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

    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

    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.

    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.

    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

    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.

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