MINICHROMOSOME MICROTUBULE BINDING ASSAY

互联网2013-09-05

1354

Determine the OD₆₀₀ and correlate the cell density from the chart. Set up four 100mL YPD cultures at the following densities: 0.7x10⁵ , 1x10⁵ , and 3.0x10⁵ cells/mL

GROWTH OF CELLS

Grow 100mL of cells to OD₆₀₀ =0.7-0.8 at 23^o C.

Add 5-10ul of BME, 15 minutes before spin.

Harvest cells in two 50mL conical tubes, spin 1.5-2.0K for 8-10min.

SPHEROPLASTING. Resuspend cells in 4mL YWB.
~undefinedNOTE: If cells have been arrested with nocodazole or alpha factor, include the inhibitor in the YWB. Let sit at room temperature for 2-3 min. Add oxalyticase [12uL 1mg/ml stock]. Incubate at 23^o C with gentle shaking to form spheroplasts. Spheroplasting should be complete within one hour; avoid incubations longer than 1h, 15min

Transfer spheroplasted cells to a TOMY tube. Spin at 3.5K for 7min. Resuspend cells in 4mL YWB (containing 5% glycerol and 1mM PMSF [0.2M stock in 100% EtOH]) by gently pipetting up and down, using a 1mL pipet. Spin at 3.5K for 7min.

Gently resuspend cells in 2.5mL 1x EBB (containing 5% glycerol and 0.4mM PMSF).

Allow cells to "swell" for 10min. at room temperature, then transfer to homogenizer. [Rinse dounce with H₂ O, EtOH to clean. Rinse dounce with EBB before using.] Homogenize cells with 5 pestle strokes (up and down is one stroke). Avoid introducing air. Transfer to clean TOMY tube.

Add 150uL 5M NaCl (final concentration ~0.3M). Incubate at room temperature for 5 min.

Add 5mL 1x EBB-plus (containing 5% glycerol, 0.1mM DTT and 150 ug/mL BSA). Thus, final concentrations in 7.5mL lysate are 0.1M NaCl. Incubate at room temperature for 45 min.

Meanwhile, thaw microtubule "seeds" and put at 37^o C for 30min. Add taxol to 10uM (e.g. 1ul 0.13mM taxol to 15ul MT seeds) and incubate an additional 15min.

Remove 250ul sample for TOTAL MATERIAL ("1T"). Keep this tube at room temperature, soas to be comparable to the other samples. Divide remaining lysate between eppendorfs tubes.

CLARIFY LYSATES

Spin eppendorf tubes at 15K for 20min. Pool supernatants in a 15mL conical tube and mix gently. Aliquot lysate to microfuge tubes (between 800ul and 1ml). Spin microfuge tubes at 15K for 20min.

Remove 250ul TOTAL SUPERNATANT ("1TS") to a fresh tube.

Remove 500ul clarified lysate from each microfuge tube to a fresh tube. Add 5ul 1mM taxol (in DMSO) to each tube - final concentration is 10uM taxol. Mix gently.

For a standard titration of binding activity, add microtubules to each tube in decreasing amount. Normally, 8, 4, 2, 1, 0.5 and 0uls (MTs made from ~3-6mg/ml PC bovine tubulin). For 0.5ul, make a 10-fold dilution of MTs in BRB80/30% glycerol buffer (containing 10uM taxol). Allow binding to proceed at room temperature for 15 min.

Pellet microtubule/minichromosome mix at 15K for 8 min. It should be possible to see the larger MT pellets.

Remove 250ul of each supernatant to fresh tube. These are the SUPERNATANT FRACTIONS. Aspirate all residual liquid with drawn out Pasteur pipets.

Resuspend MT pellets in 250ul of 1x EBB (containing 0.1mM DTT, 100mM NaCl, 5% glycerol and 1mg/ml BSA, but NO PMSF!). These are the PELLET FRACTIONS. NOTE: Ultimately, PELLETs will be 2x concentrated relative to SUPs.

Add 250ul of 2x ASSAULT buffer (containing tRNA and øX174) to all samples. Remove protein by adding 20uls of Proteinase K solution (15mg/ml from Boehringer Mannheim) to each tube. Incubate tubes at 50^o C for ~1-1.5h (longer is better).

After Proteinase K treament, precipitate DNA by adding 250ul 6M NH₄ OAc and 700ml isopropanol. Store at -20^o C overnight.

Spin down DNA at 15K, 30min. 4^o C. Carefully aspirate or decant supernatants, removing as much supernatant as possible. NOTE: The "PELLET SAMPLE" precipitated pellets are often small. Wash all DNA pellets with 100-200ul 70% ethanol. Spin briefly (~5min) and aspirate the ethanol supernatant. Allow DNA pellets to air dry. Do not invert tubes.

Resuspend all DNA pellets in 30ul TE, incubating at room temperature or 4^o C for several hours.

PREPARATION OF SAMPLES FOR SOUTHERN ANALYSIS.

Remove 15ul of each sample to a fresh tube. Add 1ul DNase-free RNase (Boehringer Mannheim, 1:1000 dilution of 2U/ul stock) to 1T, 1Ts, and all SUPERNATANT fraction. The PELLET fractions do not have to be "RNased". Incubate at room temperature for at least 30min. Add sample buffer to all tubes.

Load samples onto a 0.6% agarose gel containing EtBr at 0.2ug/ml. Include a small amount of supercoiled test plasmid (e.g. pDK370) in the DNA size standards to serve as a positive control for hybridization. The final concentration of supercoiled plasmid should be such that ~0.1ng is loaded. Run gel at 20 or 30V overnight.

Photograph gel. Note whether the intensities of the øX174 bands are even in all lanes and whether 2u circle DNA is visible in the SUPERNATANT fractions.

Process gel for transfer to GeneScreen Plus according to the Posiblot protocol. Transfer for at least 90 min. UV crosslink DNA to membrane. Air dry.

HYBRIDIZATION.

Prehybridize blot at 65^o C for ~3h in Church buffer containing 0.5mg/ml denature salmon sperm DNA (usually 14ml Church buffer plus 0.7ml 10mg/ml SS DNA per blot). Add probe 10⁵ cpms per lane) and hybridize at 65^o C overnight (at least 18h). Wash blot 2x with Buffer 1, 15min each at 65^o C and 2x with Buffer 2, 15min each at 65^o C. Monitor the blot--the last wash may not be necessary. NOTE: Use the 0.9kb SmaI/PstI URA3 fragment from pDK377 to visualize pDK370 or other URA3-containing plasmids. Use the 1.8kb SalI/ClaI LEU2 fragment from pDK255 to visualize YCp41 or other LEU2-containing plasmids. Use the ~2kb XhoI/SalI fragment from CV13 to visualize endogenous 2ucircle DNA. Note: make up the YWB, EBB + 2X assault buffer fresh each experiment.

YWB per 10ml 5mL 2M Sorbitol (if NZ arrested, add 40uL 1.5mg/mL 0.336mL 1M K₂ HPO₄ N2 to 4mL YWB) 0.064mL 1M KH₂ PO₄ 4.6mL dH₂ O	YWB, glycerol, PMSF 5mL 2M Sorbitol 0.336mL 1M K₂ HPO₄ 0.064mL 1M KH₂ PO₄ 0.5mL 100% ultrapure glycerol 0.05mL 0.2M PMSF 4.05mL dH₂ O
1X EBB, glycerol, PMSF (5mL) 1mL 5X EBB 0.25mL 100% glycerol 12.5uL 0.2M PMSF 3.75uL dH₂ O	1X EBB, glycerol, DTT, BSA (10mL) 2mL 5X EBB 0.5mL glycerol 10uL 100mg/mL BSA 10uL 0.1M DTT 7.48mL dH₂ O
1X EBB, DTT, NaCl, BSA 5mL 1mL 5X Ebb 5uL 0.1M DTT 0.1mL 5M NaCl 50uL 100mg/mL BSA 0.25mL glycerol 3.6mL dH₂ O 2mL EBB 0.2mL NaCl 0.1 BSA

REAGENTS
YWB: 50mL 2M sorbitol 16.8mL 0.2M K₂ HPO₄ 3.2mL 0.2M KH₂ PO₄ 30mL H₂ O	0.1M DTT
0.2M PMSF in ethanol or ispropanol	Glusulase
5X EBB : 5mL 1M MgCl₂ 5mL TRIS-HCl, pH7.4 0.1mL 0.5M EDTA 90mL H₂ O	5M NaCl
1mM taxol in DMSO	2X ASSAULT BUFFER: 5mL 10% SDS, UltraPure 5mL 0.5M EDTA 5mL HEPES-KOH, pH 7.6 [7.5 w/NaOH] 85mL H₂ O ~undefined2X_A.B._can_be_frozen_in_10mL_aliquots.~Kbr_~H~M~2~1~0~0~0~0~0~P3.5mL_Assault_buffer~E_0.5mL_tRNA~HøX174]
tRNA/øX174 DNA: 1mL 1mg/mL tRNA in H₂ O 0.1mL 10 ug/mL uncut øX174 DNA	5X BRB80 100mLs 5X stock 80mM Pipes pH6.8(KOH) 10mL 800mM Pipes pH6.8 (KOH) 1mM EGTA 1ml 100mM EGTA 1mM MgCl₂ 0.1mL 1M MgCl₂

MINICHROMOSOME-MICROTUBULE BINDING ASSAY

GROWTH OF CELLS. Grow 100mL of cells to OD₆₀₀ =0.7-0.8 at 23^o C. For good binding activity it has proved important to maintain cells in log phase. Normally, 2 days prior to day of experiment, a single medium-sized cology is picked from selective medium and inoculated into 5mL YPS (or -URA, as I do). Two additional dilutions are made from this "neat" inoculum (e.g. 1:10 and 1:25). The goal is to have late log phase cultures the next day (~4x10⁷ cells/mL).

The day prior to experiment, make a 1:10 dilution of the suitable 5mL overnight. Determine the OD₆₀₀ and correlating cell density from the chart. Set up three 100mL YPD cultures at the following densities: ~0.7x10⁵ , 1x10⁵ and 1.5x10⁵ cells/mL.

Harvest cells in two 50mL conical tubes, 1.5-2.0K for 8-10 min. Wash with H₂ O and consolidate cells in one tube. Re-spin. NOTE: If cells have been arrested with nocodazole or a factor, include the inhibitor in the H2O wash.

SPHEROPLASTING.

Re-suspend cells in 4mL YWB (containing 10mM beta-mercaptoethanol and 1mM PMSF). Le sit at room temperature for 2-3 min. Add 100 uL glusulase. Incubate at 23^o C with gentle shaking to form spheroplasts. Spheroplsting should be complete within one hour; avoid incubations longer than 1h, 15min. NOTE: If cells have been arrested with nocodaole or alpha factor, include the inhibitor in the YWB.

Transfer spheroplasted cells to a TOMY tube. Spin at 3.5K for 7min. Re-suspend cells in 4mL YWB (containing 1mM PMSF only) by gently pipetting up and down, using a 1mL plastic pipet. Re-spin. Repeat wash process, for a total of two washes. NOTE: If cells have been arrested with nocodazole or alpha factor, include the inhibitor in the FIRST WASH ONLY.

Gently reuspend cells in 2.5mL 1X EBB (containing 0.1mM DTT and 0.5mM PMSF). Transfer to homogenizer.

Allow cells to "swell" for 10min at room temperature, then homogenize cells with 5 pestle strokes (up and down is one stroke). Avoid introducing air. Transfer to clean TOMY tube.

Add 150uL 5M NaCl (final concentration ~0.3M). Incubate at room temperature for 5min.

Add 5mL 1X EBB-plus (containing 0.1mM DTT, 150 ug/mL BSA, and 0.5mM PMSF). Thus, final concentrations in 7.5mL lysate are 0.1M NaCl and 100ug/mL BSA. Incubate at room temperature for 45 min.

Thaw microtubule "seeds" and put at 37^o C for 30min. Add taxol to 10uM (e.g. 1uL 0.13mM taxol to 12 uL MTs) and incubate an additional 15min.

Remove 250uL sample for TOTAL MATERIAL (so-called "1T"). Keep this tube (and 1Ts, see below) at room temperature, so as to be comparable to the othe rsamples. Divide remaining lysate between two TOMY tubes.

CLARIFY LYSATES.

Spin TOMY tubes at 15K for 20min. Pool supernatants in a 10mL conical tube and mix gently. Aliquot lysate to microfuge tubes. The minimum number of tubes is [1 + the number of binding reactions planned] (usually 0.7-1mL extract per tube). Spin microfuge tubes at 15K for 20min.

Remove 250uL STARTING MATERIAL ("1TS") to a fresh tube.

Remove 500uL clarified lysate from each microfuge tube to a fresh tube. Add 5uL 1mM taxol (in DMSO) to each tube - final concentration is 10uM taxol. Mix gently.

For a standard titration of binding activity, add microtubules to each tube in increasing amount. Normally, 0, 0.2, 0.5, 1, 2, and 4uL For 0.2uL and 0.5uL, make a 10-fold dilution of MTs in BRB80/30% glycerol buffer (containing 10uM taxol). Allow binding to proceed at room temperature for 15min.

Pellet MTs (and associated minichromosomes) at 15K for 8min. It should be possible to see the larger MT pellets (usually bluish-white).

Remove 250uL of each supernatant to fresh tube. These are the SUPERNATANT FRACTIONS. Aspirate all residual liquid with drawn out Pasteur pipets.

Resuspend MT pellets in 250uL of 1X EBB (containing 0.1mM DTT, 100mM NaCl, and 1mg/ml BSA, but NO PMSF!). These are the PELLET FRACTIONS. NOTE: Ultimately, PELLETs will be 2X concentrated relative to SUPs.

Add 250uL of 2X ASSAULT buffer (containing tRNA and øX174 DNA) to all samples (i.e. all sups and pellets, as well as 1T and 1Ts). Remove protein by adding 5mL of Proteinase K solution (20mg/mL from Boehringer Mannheim) to each tube. Incubate tubes at 50^o C for ~1-1.5h. (longer is better).

After Proteinase K treatment, precipitate DNA by adding 250uL 6M NH₄ OAc and 500uL isopropanol. Store at -20^o C overnight.

Spin down DNA in TOMY at 15K, 30min. 4^o C. Carefully aspirate or decant supernatants, removing as much supernatant as possible. NOTE: The "PELLET" pellets are often small. Wash all DNA pellets with 100-200 uL 70% ethanol.Spin briefly (~5min.) and aspirate the ethanol supernatant. Allow DNA pellets to air dry. Do not invert tubes.

Resuspend all DNA pellets in 30uL TE, incubating at room temperature or 4^o C for several hours.

PREPARATION OF SAMPLES FOR SOUTHERN ANALYSIS.

Remove 15uL of each sample to a fresh tube. Add 1uL DNase-free Rnase (Boehringer Mannheim, 1:1000 dilution of 2U/uL stock) to 1T, 1Ts, and all SUPERNATANT fractions. The PELLET fractions do not have to be "RNased". Incubate at room temperature for at least 30min. Add sample buffer to all tubes.

Load samples onto a 0.6% agarose gel containing EtBr at 0.2ug/mL. Include a small amount of supercoiled test plasmid (e.g. pDK370) in the DNA size standards to serve as a positive control for hybridization. The final concentration of supercoiled plasmid should be such that ~0.1ng is loaded. Run gel at 20 or 30V overnight.

Photograph gel. Note whether the intensitites of the øX174 bands are even in all lanes and whether 2u circle DNA is visible in the SUPERNATANT (hopefully) fractions.

Process gel for transfer to GeneScreen Plus according to the Posiblot protocol. Transfer for at least 90 min. UV crosslink DNA to membrane. Air dry.

HYBRIDIZATION.

Prehybridize blot at 65^o C for ~3 h. in Church buffer containing 0.5 mg/mL denatured salmon sperm DNA (usually 14mL Church buffer plus 0.7mL 10 mg/mL SS DNA per blot). Add probe (10⁵ cpms per lane) and hybridizae at 65^o C overnight (at least 18h.). Wash blot 2X with Buffer 1, 15 min. each at 65^o C and 2X with Buffer 2, 15min. each at 65^o C. Monitor the blot - the last wash may not be necessary.

NOTE: Use the 0.9kb Sma1/Pst1 URA3 fragment from pDK377 to visualize pDK370 or other URA3-containing plasmids. Use the 1.8kb Sal1/Cla1 LEU2 fragment from pDK255 to visualize YCp41 or other LEU2-containing plasminds. Use the ~2kb Xho1/Sal1 fragment from CV13 to visualize endogenous 2u circle DNA.

YWB for spheroplasting: 5mL YWB 25uL 0.2M PMSF 3.5uL beta-ME	YWB for wash: 8mL YWB 40uL 0.2M PMSF
1X EBB (to break): 2mL 5X EBB 25uL 0.2M PMSF 10uL 0.1M DTT 8mL H₂ O	1X EBB-plus (to dilute): 1mL 5X EBB 12.5uL 0.2M PMSF 5uL 0.1M DTT 75uL 100mg/ml BSA 4mL H₂ O
1X EBB-double plus (for MT pellets): 1mL 5X EBB 12.5uL 0.2M PMSF 5uL 0.1M DTT 50uL 100mg/ml BSA 100uL 5M NaCl 3.9mL H₂ O	2X Assault Buffer: X mL 2X ASSAULT BUFFER (X=0.25mL x #samples) 0.1 x XmL tRNA/øX174 DNA
3.75 dH₂ O	0.25 EDTA
0.25 SDS	0.25 HEPES
0.5 tRNA + øX174