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Nucleosome Reconstitution 1,Expression and Purification of Recombinant. Histones and Nucleosome Reconstitution, Karolin Luger Thomas J Rechsteiner and Timothy J Richmond. 1 Introduction, In vitro studies on nucleosome core particles NCPs and nucleosomes have. generally been limited to the use of histone proteins isolated from chromatin. Numerous reliable and well established methods have been described of. obtaining single histone proteins in significant quantity e g refs 1 and 2 and. references therein Briefly the histone complexes histone octamer or his. tone tetramer and histone dimer are isolated from long chromatin which is. extracted from nuclei The histone complexes can be further fractionated into. individual histone proteins This approach suffers from several disadvantages. First the procedure is time consuming and depends on the availability of fresh. tissue or blood from the organism of choice Second histone proteins isolated. from natural sources are often degraded by contaminating proteases 3 Third. histone isotypes and posttranslational modifications of histone proteins give. rise to heterogeneity The extent of heterogeneity and modification strongly. depend on the type and developmental state of the tissue from which chromatin. is isolated and can vary significantly between different batches Fourth and. most important only naturally occurring histone proteins can be obtained by. this method, The availability of large amounts of naturally occurring mutants or of new. site directed mutants of the highly conserved histone proteins will be. extremely valuable in our attempts to reconcile the observed functions and. biophysical properties of the NCP with the recently determined atomic struc. ture 4 The ability to express all four histone proteins in bacteria has allowed. us to develop a method for the mapping of nucleosome position to base pair. From Methods in Molecular Biology Vol 119 Chromatin Protocols. Edited by P B Becker Humana Press Inc Totowa NJ,2 Luger Rechsteiner and Richmond.
resolution 5 and has been instrumental in the structure determination of the. NCP at high resolution 4 In comparison to yeast expression systems yields. are high protease activity is low and purification does not rely on the presence. of histidine tags or other fusions 6 7, This section describes the overexpression of histones H2A H2B H3 and. H4 both as full length proteins and corresponding trypsin resistant globular. domains as defined in 8 A simple and efficient purification protocol yields. large amounts of homogenous protein in denatured form The methods for. refolding and purification of histone octamer and for assembly and purifica. tion of nucleosome core particles using 146 bp of DNA are described together. with a protocol for a high resolution gel shift assay to monitor the purity and. homogeneity of the final core particle preparation. 2 Materials,2 1 Histone Expression, 1 pET histone expression plasmids 2 and transformation competent cells of the. expression strain BL21 DE3 pLysS 9, 2 2X TY AC media 16 w v bacto tryptone 10 w v yeast extract and 5 w v. NaCl supplemented with 100 g L ampicillin and 25 g L chloramphenicol. 3 AC agar plates 10 w v bacto tryptone 5 w v yeast extract 8 w v NaCl. and 1 5 w v Agar supplemented with 100 g L ampicillin and 25 g L. chloramphenicol, 4 IPTG 0 4M Isopropyl D thiogalactopyranoside in water pass through 0 2 m. sterile filter store frozen in aliquots, 5 Wash buffer 50 mM Tris HCl pH 7 5 100 mM NaCl 1 mM Na EDTA 1 mM.
benzamidine Shortly before use add 5 mM 2 mercaptoethanol. 2 2 Histone Purification,1 Wash buffer as in Subheading 2 1 item 5. 2 TW buffer wash buffer with 1 v v Triton X 100, 3 Unfolding buffer 7M guanidinium HCl 20 mM Tris HCl pH 7 5 10 mM DTT. Pass through 0 4 m filters before use, 4 Amberlite MB3 or similar ion exchange resin for batch deionization of urea stock. 5 SAU 1000 7M urea deionized 20 mM sodium acetate pH 5 2 1 M NaCl 5 mM. 2 mercaptoethanol 1 mM Na EDTA Pass through 0 4 m filters before use. 6 SAU 200 7M urea deionized 20 mM sodium acetate pH 5 2 0 2M NaCl 5 mM. 2 mercaptoethanol 1 mM Na EDTA Pass through 0 4 m filters before use. 7 SAU 600 7M urea deionized 20 mM sodium acetate pH 5 2 0 6M NaCl 5 mM. 2 mercaptoethanol 1 mM Na EDTA Pass through 0 4 m filters before use. 8 Gel filtration column XK 50 Pharmacia Uppsala Sweden packed with. Sephacryl S 200 high resolution gel filtration resin Pharmacia Gel bed 5 cm. diameter 75 cm height,Nucleosome Reconstitution 3, 9 An HPLC system equipped with a TSK SP 5PW HPLC column 2 15 cm 15 0 cm. Toyo Soda Manufacturing Company Tokyo Japan, 10 Dialysis tubing molecular weight cutoff 6 8 kDa widths 5 cm and 2 5 cm Prepare.
according to the supplier and rinse thoroughly with distilled water before use. 11 Standard SDS PAGE equipment 18 SDS gels for analysis of protein fractions 10. 2 3 Histone Octamer Reconstitution,1 Unfolding buffer as in Subheading 2 2 3. 2 Refolding buffer 2M NaCl 10 mM Tris HCl pH 7 5 1 mM Na EDTA 5 mM. 2 mercaptoethanol, 3 Gel filtration column HiLoad 16 60 Superdex 200 prep grade Pharmacia. equipped with UV detector and fraction collector at 4 C see Note 7. 4 Standard SDS PAGE equipment see Subheading 2 2 step 11. 5 Concentration device suitable for 1 25 mL volumes e g Sartorius ultrathimble. Sartorius AG G ttingen Germany or Centricon 10 Amicon AG Beverley MA. 2 4 Nucleosome Core Particle Reconstitution, 1 Purified histone octamer at a concentration of at least 0 75 mg mL in refolding buffer. 2 DNA of length greater than 138 bp with a known concentration at least 3 mg mL. 3 A peristaltic pump with a double pump head capable of maintaining a flow rate. of approx 2 6 mL min e g Gilson Minipuls 3 peristaltic pump equipped with. tubing with 2 5 mm inner diameter Gilson Medical Electronics SA Villier leBel. France or two peristaltic pumps, 4 A reconstitution flask with connected tubing as shown in Fig 1. 5 Buffers for reconstitution, RB high 2M KCl 10 mM Tris HCl pH 7 5 1 mM EDTA 1 mM DTT.
RB low 0 25M KCl 10 mM Tris HCl pH 7 5 1 mM EDTA 1 mM DTT. 2 5 Nucleosome Core Particle Purification, 2 5 1 Purification by HPLC Ion Exchange Chromatography. 1 TES 250 0 25M KCl 10 mM Tris HCl pH 7 5 0 5 mM EDTA. 2 TES 600 0 6M KCl 10 mM Tris HCl pH 7 5 0 5 mM EDTA. 3 A HPLC apparatus equipped with a TSK DEAE 5PW HPLC column 2 15 15 0 cm. or a TSK DEAE 5PW HPLC column 7 5 75 mm Toyo Soda Manufacturing. preferrably at 4 C, 2 5 2 Purification by Preparative Gel Electrophoresis. High Resolution Gel Shift Assay, 1 Model 491 Prep Cell Bio Rad Laboratories Richmond CA with a standard. power supply connected to a UV detector and a fraction collector and equipped. with a peristaltic pump, 2 Gel running buffer 0 20X TBE 1X TBE 89 mM Tris HCl 89 mM boric acid. and 2 5 mM EDTA,4 Luger Rechsteiner and Richmond, Fig 1 Schematic drawing of the experimental apparatus for reconstitution We use.
a 500 mL glass flask as a reconstitution vessel and a peristaltic pump with a four. channel head Standard glass tubes are bent in the appropriate manner and are con. nected by silicone tubing The reconstitution vessel contains RB high to start. 3 Acrylamide stock solution 29 5 acrylamide 0 5 bis acrylamide in water. Deionized by stirring with Amberlite MB3 and stored at 4 C. 4 Dialysis membrane molecular weight cutoff 6 8 kDa cut to a circle with a. radius of 3 cm Prepare according to the supplier and rinse thoroughly with dis. tilled water before use, 5 Concentration device as specified in Subheading 2 3 5. 6 Storage buffers TCS buffer,20 mM Tris HCl pH 7 5 1 mM EDTA 1 mM DTT. CCS buffer 20 mM K Cacodylate pH 6 0 1 mM EDTA,3 1 Histone Expression. Expression plasmids for the individual histone proteins and their N termi. nally truncated versions based on the T7 expression system 9 have been. described previously 2 High level expression of the Xenopus laevis histone. genes for H2A H2B and H3 does not necessitate adaptation of the codon. usage despite the presence of several codons with low usage in Escherichia. coli These proteins can be expressed with similar efficiencies as N terminal. Nucleosome Reconstitution 5, fusion proteins or with the coding region fused directly to the promoter In. contrast the Xenopus laevis gene for histone H4 could only be expressed after. redesigning the coding region of H4 to optimize for codon usage in E coli 2. Expression levels of histones H2A and H2B appear to be insensitive to the. sequence variant that we have expressed but histone H3 expression levels do. vary between different sequence variants or mutated genes H4 expression is. sensitive to amino acid substitutions and can drop to an undetectable level for. certain point mutations Typical yields for H2A H2B and H3 are 50 80 mg of. pure protein per liter of cell culture while yields for H4 are 4 5 times lower. see Note 1, 1 Transform BL21 DE3 pLysS cells with 0 1 1 g of the pET histone expression.
plasmid and plate on AC agar plates Incubate the plates at 37 C overnight. 2 First perform a test expression by incubating five 5 mL aliquots of 2X TY AC. each inoculated with one single colony from the agar plate Shake at 37 C for. approx 4 h or until the OD600 is between 0 3 and 0 6 Transfer 0 5 mL of the. culture into a sterile Eppendorf tube add 0 2 mL of sterile glycerol mix well and. store at 80 C this will serve as the glycerol stock for large scale expressions. Induce all but one culture by addition of IPTG to a final concentration of 0 2 mM. Leave one sample uninduced as a negative control Incubate for another 2 3 h at. 37 C harvest by centrifugation and boil the cell pellets in 100 L of protein gel. loading buffer Load 20 L per sample on an 18 SDS polyacrylamide gel SDS. PAGE and determine the culture with the maximum expression. 3 The evening before performing the large scale expression restreak the glycerol. stock for this culture on an AC agar plate and incubate at 37 C over night see. 4 The next morning inoculate five aliquots of 4 mL 2X TY AC media with one. colony from this plate and shake at 37 C for approx 4 h OD600 0 3 Use the. combined precultures to inoculate 100 mL of 2X TY AC media Shake at 37 C. for about 2 h or until the culture is slightly turbid OD600 0 4 see Note 2. 5 Inoculate 12 2 L Erlenmeyer flasks containing 500 mL 2X TY AC media with 8 mL. of the 100 mL starter culture Shake at 200 rpm and 37 C until the OD600 has. reached 0 6 this takes about 3 h see Note 2 Induce by addition of 0 2 mM. IPTG final concentration and shake for another 2 h H3 and H4 or 3 h H2A. 6 Harvest the cells by centrifugation at room temperature Resuspend homoge. neously in 100 mL wash buffer and flash freeze in liquid nitrogen see Note 3. 3 2 Histone Purification, The purification protocol involves three steps preparation of inclusion bod. ies gel filtration under denaturing conditions and HPLC ion exchange chro. matography under denaturing conditions, Analysis of the pure proteins by gel electrophoresis SDS and triton urea. acid PAGE mass spectroscopy amino acid analysis and by sequencing the. 6 Luger Rechsteiner and Richmond, N terminus using Edman degradation shows that all preparations were highly. homogenous and free of modifications Whereas the terminally truncated his. tone proteins retain the N terminal methionine residue the full length histones. are completely free of this methionine residue and begin with the native. sequence 2,3 2 1 Inclusion Body Preparation, 1 Start the equilibration of the Sephacryl S 200 gel filtration column in the morn. ing prepare 2 L of 9M urea in water Heat to dissolve and deionize with Amberlite. MB3 see Note 4 Prepare 2 L of SAU 1000 buffer Equilibrate the column with. 2 L of filtered and degassed SAU 1000 buffer at a flow rate of 3 mL min. 2 To avoid overloading of the gel filtration column no more than the equivalent of. 6 L of cell culture should be processed at one time For histones with moderate. expression e g all H4 variants up to 12 L of culture can be used Thaw the cell. suspension in a warm water bath The cell suspension will become extremely. viscous as lysis occurs Stir occasionally until completely thawed 20 30 min. Transfer into a wide short measuring cylinder and adjust the volume with wash. buffer to 150 mL Reduce viscosity by shearing with a Turrax stirrer Check. whether the mixture is still viscous by using a pasteur pipet and if necessary. repeat shearing step Centrifuge immediately for 20 min at 4 C and 23 000g The. pellet contains inclusion bodies of the corresponding histone protein. 3 Resuspend the pellet completely in 150 mL TW buffer using a 10 mL plastic. pipet Spin for 15 min at 4 C at 12 000 rpm Repeat this step twice with TW. buffer and twice with wash buffer see Note 5 After the last wash the drained. pellet can be stored at 20 C until further processing. 4 With a spatula transfer the pellet to a 50 mL centrifuge tube Add 1 mL of DMSO. and soak the pellet for 30 min at room temperature Mince the pellet with a spatula. Slowly add 40 mL of unfolding buffer and stir gently for 1 h at room temperature. The pellet should eventually almost completely dissolve see Note 4. 5 Remove cell debris by centrifugation at 20 C and 23 000g The supernatant con. tains the unfolded proteins Reextract the pellet with 10 mL of unfolding buffer. and combine the supernatants,3 2 2 Gel Filtration, 1 Load a maximum of 60 mL of the sample on the equilibrated S 200 column at a.
flow rate of 3 mL min Record the elution profile at a wavelength of 280 nm and. collect fractions of an appropriate size, 2 Analyze peak fractions by 18 SDS PAGE The first peak will contain DNA and. larger proteins but can be merged with the histone peak Because of the high dilu. tion factor and the small molar absorption of histones the histone peak is often. small and unobtrusive Pool fractions containing histone proteins see Note 6. 3 The protein is dialyzed thoroughly against at least three changes of distilled water. containing 2 mM 2 mercaptoethanol at 4 C Dialysis bags with a cutoff of 6 8. kDa are sufficient even for the globular domain of H4 but leave enough room for. Nucleosome Reconstitution 7, Molecular Weights and Molar Extinction Coefficients for Full Length. and Trypsin Resistant Globular Domains of Histone Proteinsa. Full length protein Globular domains,Histone Mol wt 276 nm Amino acid Mol wt 276 nm. H2A 13 960 4050 19 118 11 862 4050,H2B 13 774 6070 27 122 11 288 6070. H3 15 273 4040 27 135 12 653 4040,H4 11 236 5400 20 102 9 521 5040.
a was calculated according to Gill and von Hippel 14 The molecular weights were deter. mined by a summation of amino acids and were confirmed by mass spectrometry 2. the volume increase Determine the concentration of the dialyzed sample using the. molecular extinction coefficients listed in Table 1 Lyophilize and store at 20 C. 3 2 3 Purification by Ion Exchange Chromatography SP 5PW. 1 Dissolve lyophilized histone protein in SAU 200 and remove insoluble matter by. centrifugation Equilibrate the preparative SP 5PW HPLC column with SAU. 200 buffer Inject a maximum of 15 mg of protein for each run Using a flow rate. of 4 mL min elute proteins with the gradients given in Table 2 buffer A SAU. 200 buffer B SAU 600, 2 Analyze the peak fractions by SDS PAGE Pool the fractions containing pure histone. protein dialyze against water as described under Subheading 3 2 2 3 and lyophilize. 3 Dissolve in a small volume of water determine the concentration using the val. ues given in Table 1 and lyophilize in Nunc vials in aliquots suitable for subse. quent octamer refolding reactions see Subheading 3 3 1 For example 4 5 mg. H3 3 5 mg H4 4 0 mg H2A 4 0 mg H2B The purified histones can be stored at. 20 C for an unlimited length of time,3 3 Refolding of the Histone Octamer. The protocol below is valid for the refolding of histone octamers from lyo. philized recombinant histone proteins All combinations of recombinant. Xenopus laevis full length and globular domain histone proteins have been. refolded to functional histone octamers The method works best for 6 15 mg. of total protein For smaller amounts of protein scale down the gel filtration. column see Note 7, 1 Dissolve each histone aliquot to a concentration of approximately 2 mg mL in. unfolding buffer Use a Pasteur pipet to ensure protein sticking to the sides of the. tube is dissolved Do not vortex Unfolding should be allowed to proceed for at. least 30 min and for no more than 3 h see Note 4,8 Luger Rechsteiner and Richmond. Salt Gradients for Elution of Histone Proteins,from an SP Columna.
H2A H2B H3 H4,t min B t min B t min B,0 0 0 0 0 0,10 0 3 30 5 50. 11 40 8 30 10 59,46 100 40 100 40 100,60 100 50 100 50 100. 61 0 51 0 51 0, aSalt gradients for elution of histone proteins from an SP 5PW HPLC. column 2 15 15 0 cm the flow rate is 4 mL min buffer A is SAUDE 200. buffer B is SAUDE 600, 2 Determine the concentration of the unfolded histone proteins by measuring OD276. Table 1 of the undiluted solution against unfolding buffer remove solid matter. by centrifugation if necessary, 3 Mix the four histone proteins to equimolar ratios and adjust to a total final protein.
concentration of 1 mg mL using unfolding buffer, 4 Dialyze at 4 C against at least three changes of 2 L of refolding buffer for 15 mg. setup use dialysis bags with a flat width of 2 5 cm The second or third dialysis. step should be performed overnight Octamer should always be kept at 0 4 C. 5 Remove precipitated protein by centrifugation There should be almost no pre. cipitate in the ideal refolding reaction Concentrate to a final volume of 1 mL. 6 Gel filtration is performed at 4 C at a flow rate of 1 mL min Load a maximum. of 1 5 mL or 15 mg of the concentrated histone octamer on the Superdex 200 gel. filtration column previously equilibrated with refolding buffer High molecular. weight aggregates will elute after about 45 mL histone octamer at 65 68 mL. and histone H2A H2B dimer at 84 mL see Note 7, 7 Check the purity and stoichiometry of the fractions on an 18 SDS PAGE Dilute. by a factor of at least 2 5 before loading onto the gel to reduce distortion of the. bands resulting from the high salt concentration Pool fractions that contain. equimolar amounts of the histone proteins, 8 Determine the concentration A276 0 45 for a solution of 1 mg mL Use for. nucleosome core particle reconstitution with DNA immediately or concentrate to. 3 15 mg mL adjust to 50 v v glycerol and store at 20 C. 3 4 Reconstitution of Nucleosome Core Particles, Specific DNA fragments of the desired length or sequence can be obtained. by a number of methods e g 11 12 Reconstitution of histone octamer with. DNA is accomplished using a modification of the salt gradient method. Nucleosome Reconstitution 9, described by Thomas and Butler 13 Briefly octamer and DNA is mixed at 2 M.
KCl and the salt concentration is reduced by dialysis to 0 25 M KCl over a. period of 36 h 12 The procedure works equally well for large up to 10 mg. and small 0 1 mg amounts of nucleosome core particles Multiple setups can. be dialyzed in one vessel If smaller amounts need to be reconstituted use. dialysis buttons e g Hampton Research Laguna Hills CA or the apparatus. described in Chapter 4, 1 Histone octamer is added to the DNA to a 0 9 molar ratio of octamer to DNA. with a final DNA concentration of 6 M Before adding the histone octamer see. Note 8 adjust the salt concentration of the DNA solution to 2 M using 4 M or. solid KCl and add DTT to a final concentration of 10 mM Incubate at 4 C for. 2 Prepare 400 mL RB high and 1600 mL RB low buffer and chill these to 4 C Set. up the dialysis apparatus as shown in Fig 1 Calibrate the pump to a flow rate of. 0 7 0 8 mL min, 3 Transfer the sample to a dialysis bag and start dialysis against 400 mL RB high. at 4 C under constant stirring Using the peristaltic pump continually remove. buffer from the dialysis vessel and replace with RB low Over a period of 36 h. an exponential gradient is generated see Note 9 After the gradient has finished. dialyze for at least 3 h against RB low If the samples are not further processed. within the next 24 h dialyze against CCS buffer If the core particle will be puri. fied by preparative gel electrophoresis see Subheading 3 5 2 dialyze against. TCS buffer,3 5 Purification of the Nucleosome Core Particle. Two methods are described for the purification of nucleosome core particles. from free octamer and or free DNA Both methods have been optimized for. NCP with 146 bp DNA but can easily be adjusted for nucleosomes with differ. ent length DNA The first method uses HPLC DEAE ion exchange chroma. tography 12 Free DNA elutes from the column at a higher salt concentration. than NCP and thus can be easily separated from the complex The second. method uses gel electrophoresis under nondenaturing conditions as a purifica. tion principle 2 Table 3 compares the advantages and disadvantages of the. two methods Both methods alone give rise to highly pure NCP preparations. the choice depends on available equipment and on the problem at hand Ion. exchange chromatography is suitable for large scale preparations on a routine. basis However certain modification of histone proteins such as covalently. bound heavy atoms might completely alter the binding and elution properties. of the NCP If a significant amount of material appears as a high molecular. weight band in a gel shift assay or if the particle is prone to salt dependent. dissociation the second method might be more suitable The two methods can. also be used in combination,10 Luger Rechsteiner and Richmond. Purification of Nucleosome Core Particles Comparison of Two Methods. DEAE ion exchange Preparative gel electrophoresis, Capacity Up tp 10 mg NCP per Maximum of 3 mg NCP per run.
run for a preparative,column 2 15 15 0 cm,Time Fast reliable Slow labor intensive. Purification No purification from Purification from both DNA and. higher order aggregates higher order complexes, Elution Salt elution might cause Free choice of elution buffer. dissociation, Reconstitution usually leads to a heterogeneous population of NCP with. respect to the position of the DNA on the histone octamer A simple heating. step 37 55 C for 20 180 min usually results in a uniquely positioned NCP. preparation for DNA 145 to 147 bp in length which is suitable for biochemical. studies and crystallization see Fig 3 and Note 10,3 5 1 Purification by Ion Exchange Chromatography. 1 Equilibrate the DEAE 5PW column with TES 250 at 4 C Centrifuge the recon. stitution mixture in Eppendorf tubes at 4 C and inject the supernatant on the. column a maximum of 10 mg core particle Samples can either be in RB low. TCS buffer or CCS buffer Using a flow rate of 4 mL min develop the column. with the gradient given in Table 4 buffer A TES 250 buffer B TES 600. while monitoring the eluent at 260 nm Adjust gradient for core particles contain. ing longer DNA fragments or different temperatures and salts NaCl instead of. KCl A typical chromatogram is shown in Fig 2A see Note 10. 2 Analyze the peak fractions by non denaturing gel electrophoresis if necessary. see Subheading 3 6 and Note 10, 3 Pool the peak fractions and immediately dialyze against three changes of TCS.
buffer at 4 C Concentrate and store on ice until use For prolonged storage dia. lyze against CCS buffer or add 5 mM potassium cacodylate at an appropriate pH. to prevent microbial growth Determine the concentration of NCP preparations. by measuring the absorbency at 260 nm of a 200 to 500 fold dilution The yield. of the above reconstitution and purification method may vary between 20 and. 80 of the DNA added to the reconstitution mixture, 3 5 2 Purification by Preparative Gel Electrophoresis. see also Subheading 3 6, 1 Prepare 20 mL of a 5 polyacrylamide gel ratio of acrylamide to bis acrylamide. 60 1 containing 0 2X TBE and pour a cylindrical gel with an outer radius of 28 mm. Nucleosome Reconstitution 11,Salt Gradient for Elution. of NCP from a DEAE 5PW,HPLC Columna,a The flow rate is 4 mL min. buffer A is TES 250 buffer B is, an inner radius of 19 mm and a height of 50 mm Polymerize and assemble.
according to instructions given in the manual for the Model 491 Prep Cell Prerun. under constant recircularization of the buffer for 90 min in 0 25X TBE at 4 C and. 10 W Record a base line at 260 nm using TCS as elution buffer see Notes. 2 After reconstitution dialyze NCP against TCS buffer and concentrate A maxi. mum of 600 L or 3 mg is mixed with sucrose to a final concentration of 5 v v. and loaded on the preparative gel Electrophorese at 10 W and elute the complex. at a flow rate of 0 7 mL min with TCS buffer Recirculate buffer Record elution at. an OD of 260 nm and collect fractions of appropriate size usually 0 7 1 0 mL. Free DNA will appear first followed by pure NCP and finally by higher. molecular weight aggregates Fig 2B see Note 11, 3 6 High Resolution Gel Shift Assay for Nucleosome Core Particles. The protocol given next routinely allows for the separation of NCP with. different translational setting of the DNA by only 10 bp Fig 3 see refs 2 5. We have observed that the ratio between acrylamide and bis acrylamide can. completely alter the relative mobilities of different NCP species with respect to. the DNA size marker 2 The choice of gel buffer also has minor effects on the. resolution of the different NCP species, 1 Prepare a native gel with the dimensions 20 20 0 1 cm using a 10 to 16 well. comb The gel material is 5 acrylamide with 60 1 acrylamide to bis acrylamide. with 0 2X TBE, 2 Prerun gels for at least 3 h at 4 C and 200 V while constantly recycling the. running buffer see Note 12,12 Luger Rechsteiner and Richmond. Fig 2 Purification of reconstituted nucleosome core particle by A ion exchange. chromatography or B preparative gel electrophoresis Elution is monitored by the. absorbance at 260 nm Separation conditions are as given in the text The approxi. mate elution times are shown for each peak but they are dependent on the flow rate. and the geometry of the set up A Purification by HPLC ion exchange chromatogra. phy NCPs with different rotational positions elute at different times from the column. The relative ratio between the main and minor peaks can vary Usually major and. minor peaks are combined additional peaks that usually exhibit baseline separation. not shown are not included In some cases free octamer can be observed to elute in. the beginning of the gradient not shown B Purification by preparative. nondenaturing gel electrophoresis Note that the three bands observed in Fig 3 cannot. be discerned by this method Faster elution will improve the separation of the peaks. but will also yield a more dilute sample, 3 Rinse slots well with 0 20X TBE shortly before loading samples Load 1 2 pmol.
of core particle solution containing 5 v v sucrose in no more than 10 L. Samples can be supplemented with bromophenol blue for easier handling. 4 Run the gel for a suitable length of time or until bromophenol blue has reached. the bottom of the gel Recycle the running buffer at all times see Note 12. Nucleosome Reconstitution 13, Fig 3 Nondenaturing gel electrophoresis of NCP reveals multiple positions of the. histone octamer on the DNA Lanes 1 3 purified NCP reconstituted from recombinant. full length histone proteins and a 146 bp fragment derived from the 5S RNA gene of. Lytechinus variegatus Before lanes 1 and 3 and after lane 2 heating for 1 h at 37 C. Lanes 4 and 5 purified NCP prepared from recombinant full length histone proteins and. a 146 bp palindromic DNA fragment derived from human alpha satellite DNA before. lane 4 and after lane 5 heating for 2 h at 37 C Note that NCP containing the asym. metric 5S RNA DNA fragment reconstitutes in two off centered and one centered rota. tional position marked with o and c respectively 5 whereas NCP containing a. palindromic sequence reconstitutes mainly to the off centered positions Nucleosomes. in the two off centered positions cannot be distinguished on the gel because the sequence. symmetry results in an identical exit angle of the ends of the DNA from the histone. octamer The relative positions of the DNA on the histone octamer in the three bands is. shown schematically ovals histone octamer bold line DNA. 5 Stain with ethidium bromide Note that free DNA is stained significantly better. by ethidium bromide than DNA bound to the histone octamer Subsequent stain. ing with Coomasssie brilliant blue is also possible. 1 Glycerol stocks of transformed BL21 DE3 pLys S can be kept at 80 C for at. least 2 mo If the glycerol stock has not been used for some time restreak and. perform the small scale expression test again For some difficult cases i e his. tone variants expression of histone proteins in cells lacking the pLysS plasmid. proved to be more successful T J Rechsteiner unpublished results. 2 Values obtained by measurements of the optical density at 600 nm depend on the. geometry of the spectrophotometer We use a Pharmacia Novaspec spectro. photometer Pharmacia Trial experiments should be performed for each indi. vidual histone variant to determine the optimal optical density for induction We. have observed that some point mutants exhibit quite different optima of cell. density for induction The starter culture should never be grown to densities. 14 Luger Rechsteiner and Richmond, higher than OD600 0 6 since the cells loose the ability to be induced even. after dilution, 3 Cells expressing histone proteins especially H4 are prone to lysis and should be. centrifuged at room temperature and for the same reason it is not recommended. to wash the cell pellet Resuspend cells well before freezing as this will improve. lysis upon thawing The cells can be stored at 20 C. 4 Urea in solution is in a slow equilibrium with isocyanate which can irreversibly. modify proteins Do not use urea containing solutions older than 24 h and always. deionize urea stock solutions before use Storage of protein in buffers containing. guanidinium HCl or urea for more than 24 h is not recommended. 5 If lysis by freezing and thawing is not complete additional lysis will occur upon. the addition of TW buffer and the cell suspension will become viscous again In. that case repeat shearing Two consecutive cycles of freezing and thawing of the. cell suspension can improve lysis significantly, 6 Unusually early elution of histone proteins from the Sephacryl S 200 gel filtra. tion column within the large DNA peak might result from the formation of. unspecific complexes between cellular DNA and histones The presence of 1 M. NaCl usually inhibits complex formation between cellular DNA and denatured. histones but this can occasionally remain a problem depending on the shearing. of the DNA For this reason or if the resolution of the gel filtration column is. insufficient the first fractions of the histone peak might be contaminated with. DNA Before pooling check the first few histone containing fractions by UV. spectroscopy for DNA contamination Discard if the spectrum exhibits a ratio of. OD260 OD280 1 0 since DNA and histone will form a precipitate after removal. of the salt Minor precipitate can be removed by centrifugation. 7 Other gel filtration resins of a similar separation range such as Superose 12 or. Sephacryl S 300 both from Pharmacia can also be used but give a lower resolu. tion Sephadex G 100 does not separate histone octamer from high molecular. weight aggregates and is therefore not recommended Separation between histone. octamer and excess H2A H2B dimer is better than from excess H3 H4 2 tetramer. Yields of pure histone octamer are usually between 50 and 75 Significant. amounts of octamer or high molecular weight aggregates can remain attached to. the column Clean the column with NaOH as recommended by the supplier. 8 If histone octamer from a glycerol stock is used dialyze over night against refolding. buffer and determine the concentration The required accuracy in the ratio between. histone and DNA ratio cannot be maintained if pipeted directly from the glycerol. stock Histone octamer should always be added last to the reconstitution mixture to. avoid premature mixing octamer and DNA at 2M salt concentrations. 9 Ensure that the dialysis bag can circle freely and rapidly to allow constant mixing. its contents Take care to adjust the position of the inlet and outlet tubing as. shown in Fig 1 This is important for two reasons, a Rotation of the dialysis bag is inhibited if it gets caught in the tubing.
b Uneven pump speed can result in either overflowing or running dry of the.

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General hints y Integral 4 Product Catalogue Fire Alarm Systems 2009 y V 1.0 B-HB-004EN 1 General hints This product catalogue describes the most important components ...

Eastern Trade of the Roman Empire based on Pliny the Elder ...

Eastern Trade of the Roman Empire based on Pliny the Elder

the Roman Empire Th. e fact tha tht e age of Pliny the Elder th, e 1st century A . D. was the great ag e of long-distanc trade ien the Roman Empir contributee tdo my choice. Severa eventl osf the 1st century B . C. stimulate thd e increas ien oriental trade. Hippalu discoveres th monsooed windn characteristis o thf Indiaec n Ocean.

kelas11 sej imtam - KTSP 2006 & Buku Literasi Sekolah

kelas11 sej imtam KTSP 2006 amp Buku Literasi Sekolah

/ Penulis Imtam Rus Ernawati, Nur Siwi Ismawati ; Editor Dwi Ratna Nurhajarini ; Ilustrator Suhardi. -- Jakarta : Pusat Perbukuan, Departemen Pendidikan nasional, 2009. vii, 98 hlm. : ilus. ; 28 cm. Bibliografi : hlm. 98 Indeks : hlm. 95 ISBN 978-979-068-061-6 nomor jilid lengkap) ISBN 978-979-068-070-8 1. Indonesia-Sejarah-Studi dan Pengajaran I. Judul II. Nur Siwi Ismawati III. Dwi Ratna ...