Specific protein-protein binding in many-component mixtures of proteins
蛋白纯化万金油
蛋白纯化万金油从纯化到星辰---CSH蛋白质纯化课侧记royluo ( 罗马情结)本文献给YL引子2000年5月6日，我一个人在合肥三孝口书店闲诳,希望买点参考书带出国去。
我注意到有一本翻译过来的书，书名叫做《蛋白质纯化与鉴定实验指南》。
这本书很奇怪，里面列出了很多具体实验的步骤和解释。
我很快意识到，这本书实际上是冷泉港实验室开设的一门《蛋白质纯化与鉴定》实验课的教材。
这本书深深的吸引了我，因为里面记录的实验十分经典，涵盖了蛋白质纯化中可能用到的大部分手段，并且对实验细节有十分详尽的解释。
我对这本书爱不释手，于是就买了下来, 而且当时就动了上这门课的念头。
这是一个很模糊的想法，没有想到的是，五年以后这个想法竟然实现了。
这一系列文章是我对这次上课经历的全面总结，一部分是八卦，一部分是流水账般的记叙，还有一些是从这课程学到的tricks, 最后也掺杂着自己过去做蛋白质纯化的心得。
想到哪说到哪，文字挺松散的，见笑了。
我来解释一下标题。
课程结束之后，每一个学员都有一份证书，印着Per Pura Ad Astra 几个大字。
这是拉丁语，直译就是“从纯化到星辰”。
原始的谚语是,Per Aspera Ad Astra, 意思是through suffering to renown。
所以“从纯化到星辰”实际的意思是，做好蛋白质纯化，以后就容易出人头地。
呵呵。
（二）同学和老师冷泉港的《蛋白质纯化与鉴定》培训班历史相当悠久，从1989年开始每年都开课，到今年是第十六次。
每年都是四月初开始，持续两个星期。
值得一提的是，冷泉港有相当多的培训班和会议，是一个科学家交流和学习的中心。
这是冷泉港享有盛名的最主要原因。
2004年底, 我下定决心上这门课，于是递交了申请。
二月份的时候的到消息，我被录取了，并且还有1000刀的tuition waiver。
可我还是高兴不起来，因为还得解决1600刀(学费一共2600刀)。
老板人很nice,但是他实在没钱了，所以我只好咬咬牙自己套腰包解决了，去年的退税就这样全搭进去了,欲哭无泪啊。
蛋白酶抑制剂
蛋白酶抑制剂破碎细胞提取蛋白质的同时可释放出蛋白酶，这些蛋白酶需要迅速的被抑制以保持蛋白质不被降解。
在蛋白质提取过程中，需要加入蛋白酶抑制剂以防止蛋白水解。
以下列举了5种常用的蛋白酶抑制剂和他们各自的作用特点，因为各种蛋白酶对不同蛋白质的敏感性各不相同，因此需要调整各种蛋白酶的浓度。
由于蛋白酶抑制剂在液体中的溶解度极低，尤其应注意在缓冲液中加人蛋白酶抑制剂时应充分混匀以减少蛋白酶抑制剂的沉淀。
在宝灵曼公司的目录上可查到更完整的蛋白酶和蛋白酶抑制剂表。
常用抑制剂PMSF PMSF即Phenylmethanesulfonyl fluoride，中文名为苯甲基磺酰氟。
分子式为C7H7FO2S，分子量为174.19，纯度>99%。
常用生化试剂，用于抑制蛋白酶.【配制方法】用异丙醇溶解PMSF成 1.74mg/ml（10mmol/L），分装成小份贮存于-20℃。
如有必要可配成浓度高达17.4mg/ml的贮存液（100mmol/L）。
【注意】PMSF严重损害呼吸道粘膜、眼睛及皮肤，吸入、吞进或通过皮肤吸收后有致命危险。
一旦眼睛或皮肤接触了PMSF，应立即用大量水冲洗之。
凡被PMSF污染的衣物应予丢弃。
PMSF在水溶液中不稳定。
应在使用前从贮存液中现用现加于裂解缓冲液中。
PMSF在水溶液中的活性丧失速率随pH值的升高而加快，且25℃的失活速率高于4℃。
pH值为8.0时，20μmmol/l PMSF水溶液的半寿期大约为85min，这表明将PMSF溶液调节为碱性（pH>8.6）并在室温放置数小时后，可安全地予以丢弃。
蛋白水解酶抑制剂啊实验室常用的啊主要用于组织匀浆时用!!1)抑制丝氨酸蛋白酶(如胰凝乳蛋白酶，胰蛋白酶，凝血酶)和巯基蛋白酶(如木瓜蛋白酶);2)10mg/ml溶于异丙醇中;3)在室温下可保存一年;4)工作浓度:17~174ug/ml(0.1~1.0mmol/L);5)在水液体溶液中不稳定，必须在每一分离和纯化步骤中加入新鲜的PMSF。
顺反子与顺反式效应
• As a result, each addition of a ribonucleotide is random, based on the relative concentration of the four ribonucleoside diphosphates added to the reaction mixtures. The probability of the insertion of a specific ribonucleotide is proportional to the availability of that molecule, relative to other available ri-bonucleotides.
what do we know ? DNA transcription RNA translation protein relationships are colinear nonoverlapping code (1 mutation, 1 amino acid)
12.2 The triplet Nature of the code
means Of deciphering the ribonucleotide composition of various triplets encoding specific amino acids.
• In contrast to RNA polymerase, polynucleotide phosphorylase requires no DNA template.
串联亲和层析 protocol
UNIT19.20 Strep/FLAG Tandem Affinity Purification(SF-TAP)to Study Protein InteractionsChristian Johannes Gloeckner,1Karsten Boldt,1,2and Marius Ueffing1,21Helmholtz Zentrum M¨u nchen,Neuherberg,Germany2Technical University of Munich,Munich,GermanyABSTRACTIn recent years,several methods have been developed to analyze protein-protein interac-tions under native conditions.One of them,tandem affinity purification(TAP),combinestwo affinity-purification steps to allow isolation of high-purity protein complexes.Thisunit presents a methodological workflow based on an SF-TAP tag comprising a doubletStrep-tag II and a FLAG moiety optimized for rapid as well as efficient tandem affinitypurification of native proteins and protein complexes in higher eukaryotic cells.Depend-ing on the stringency of purification conditions,SF-TAP allows both the isolation ofa single tagged-fusion protein of interest and purification of protein complexes undernative conditions.Curr.Protoc.Protein Sci.57:19.20.1-19.20.19.C 2009by John Wiley&Sons,Inc.Keywords:SF-TAP r tandem affinity purification r protein complexesINTRODUCTIONThe analysis of protein-protein interactions under native conditions has been a challengeever since immunoprecipitation(IP)became a common methodology.Low yields andnonspecific binding of proteins have been associated with IP.On the other hand,IPfacilitates targeted analysis of protein interactions with respect to a predefined proteinof interest,given that a suitable antibody is available that features monospecificity andselectivity for this protein.Tandem affinity purification(TAP;UNIT19.19)can significantly reduce the backgroundcaused by nonspecific binding of proteins,as it combines two affinity purifications basedon two different affinity matrices(Rigaut et al.,1999).TAP has been widely used topurify protein complexes from different species(Collins and Choudhary,2008).The TAPtechnique was originally developed to analyze the yeast protein interactome(Gavin et al.,2002).Although the original TAP tag,consisting of a Protein A-tag,a TEV(tobacco etchvirus)protease cleavage site,and a calmodulin binding peptide(CBP)tag,has alreadybeen successfully used in mammalian cells(Bouwmeester et al.,2004),several featuresof thisfirst-generation tag remain suboptimal,such as its high molecular mass(21kDa),the dependency on proteolytic cleavage,and CBP,which may interfere with calciumsignaling within eukaryotic cells.This unit presents an alternative TAP protocol for theisolation of protein complexes from higher eukaryotic cells.The Strep/FLAG tandemaffinity purification(SF-TAP)tag(Gloeckner et al.,2007)combines a tandem Strep-tagII(Skerra and Schmidt,2000;Junttila et al.,2005)and a FLAG tag,resulting in a small4.6-kDa tag.Both moieties have a medium affinity and avidity to their immobilizedbinding partners.Therefore,the tagged fusion proteins and their binding partners canbe recovered under native conditions without the need for time-consuming proteolyticcleavage.In thefirst step,desthiobiotin is used for elution of the SF-TAP fusion proteinfrom the Strep-Tactin matrix.In the second step,the FLAG octapeptide is used for elutionof the SF-TAP fusion protein from the anti-FLAG M2affinity matrix.An overview of the Current Protocols in Protein Science19.20.1-19.20.19,August2009Published online August2009in Wiley Interscience().DOI:10.1002/0471140864.ps1920s57Copyright C 2009John Wiley&Sons,Inc.Identification of Protein Interactions19.20.1 Supplement57Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.2Supplement 57Current Protocols in Protein Science A B 1. purification 2. purification binding to Strep-Tactin binding to FLAG matrix elution with desthiobiotin elution with FLAG peptide Key:SF-TAP desthiobiotin FLAG peptide Figure 19.20.1The S trep/FLAG ta n dem affin ity p u rificatio n .(A )N-a n d C-termi n al S F-T AP ta gs (POI,protei n of i n tere s t).(B )Overview of both p u rificatio n s tep s .(1)P u rificatio n by the ta n dem S trep-ta g II moiety:bi n di ng to S trep-T acti n matrix followed by el u tio n with de s thiobioti n .(2)P u rificatio n by the FLAG-ta g moiety:bi n di ng to a n ti-FLAG M2affin ity matrix followed by el u -tio n with FLAG peptide.Abbreviatio ns :s p.,s pecific i n teractor s (s how n a s g ray circle s );n .s p.,n o ns pecific protei ns (co n tami n a n t s ;s how n a s white circle s ).SF-TAP technique and the tag sequence is shown in Figure 19.20.1.The SF-TAP protocol represents an efficient,fast and straightforward purification of protein complexes from mammalian cells within 2hr.This unit describes the full workflow,starting with the cell culture work needed for recombinant expression of the SF-TAP fusion proteins,followed by the SF-TAP protocol (see Basic Protocol 1)and ending with mass spectrometric analysis of the samples (see Basic Protocol 4).Special focus is given to the crucial step of sample preparation for mass spectrometry.For the identification of associated proteins following SF-TAP,the volume of the SF-TAP eluates is reduced by ultrafiltration using centrifugal units with a low molecular weight cut-off or by chloroform/methanol precipitation (see Support Protocol 2).The samples are then directly subjected to proteolytic digestion (see Basic Protocol 2)for analysis on a nano liquid chromatography (LC)–coupled electron sprayIdentification of Protein Interactions 19.20.3Current Protocols in Protein Science Supplement 57Figure 19.20.2Flow chart of a S F-T AP approach i n cl u di ng M S ide n tificatio n of cop u rified pro-tei ns .Thi s figu re co nn ect s all protocol s pre s e n ted i n thi s un it.tandem mass spectrometer.For complex samples,which contain many proteins,an alternative procedure for SDS-PAGE pre-fractionation is provided,including a method for sensitive MS-compatible Coomassie protein staining (see Support Protocol 3)followed by in-gel proteolytic digestion (see Basic Protocol 3).By reducing sample complexity,pre-fractionation helps to increase the number of protein identifications on state-of-the-art LC-coupled tandem mass spectrometers.Representative MS-analysis protocols are provided for an Orbitrap mass spectrometer (Thermo Fisher Scientific),a fast and sensitive system allowing high identification rates from SF-TAP purifications even with low amounts of protein in the sample (see Basic Protocol 4).Finally,a strategy for meta analysis of mass spectrometric data sets using the Scaffold software is provided (see Support Protocol 4).It can generally be used for the analysis of large MS/MS data sets.Figure 19.20.2provides a flowchart of the entire analytical process.Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.4Supplement 57Current Protocols in Protein ScienceBASICPROTOCOL 1STREP/FLAG TANDEM AFFINITY PURIFICATION (SF-TAP)OF PROTEIN COMPLEXES FROM HEK293CELLS A flowchart of the SF-TAP procedure is shown in Figure 19.20.3.Materials HEK293cells (ATCC no.CRL-1573)Complete DMEM containing 10%FBS (APPENDIX 3C )SF-TAP vectors with appropriate insert,and empty control plasmid (see Critical Parameters)Negative control (see annotation to step 3,below)Transfection reagent of choice (see UNIT 5.10)Phosphate-buffered saline (PBS;APPENDIX 2E ),prewarmed Lysis buffer (see recipe)Strep-Tactin Superflow resin (IBA GmbH,cat.no.2-1206-10)Tris-buffered saline (TBS;see recipe)Wash buffer (see recipe)Desthiobiotin elution buffer:dilute 10×buffer E (IBA GmbH,cat.no.2-1000-025)1:10in H 2O (final concentration,2mM desthiobiotin)Anti–FLAG M2agarose (Sigma-Aldrich)FLAG elution buffer (see recipe)14-cm tissue culture plates Cell scraper Millex GP 0.22-μm syringe-driven filter units (Millipore)End-over-end rotator Microspin columns (GE Healthcare,cat.no.27-3565-01)End-over-end rotator Microcon YM-3centrifugal filter devices (Millipore)Additional reagents and equipment for transfection of mammalian cells (UNIT 5.10)Transfect HEK293cells 1.Seed HEK293cells on 14-cm plates at ∼1–2×107cells per dish in complete DMEM medium containing 10%FBS.The amount of cells used for SF-TAP purification can be varied depending on the ex-pression levels of the bait ually,four 14-cm dishes,corresponding to a final amount of ∼4×108HEK293cells,is a good starting point.Strong overexpression of the bait protein usually increases copurification of heat-shock proteins such as HSP70.For in-depth analysis,it is therefore recommended to generate cell lines stably expressing the bait protein.See Support Protocol 1for a stable transfection method.2.Grow cells overnight.3.Transfect cells with the SF-TAP plasmids using a transfection reagent of choice (according to manufacturer’s protocols).HEK293cells can be easily transfected with lipophilic transfection reagents.The trans-fection efficiency is usually >80%.For a typical SF-TAP experiment,1to 4μg plasmid per 14-cm dish is used.Depending on the cell type other transfection reagents may be favorable (also see UNIT 5.10).Although SF-TAP purifications typically exhibit low background caused by nonspecific binding of proteins to the affinity matrix,a suitable negative control should be used in every experiment.Cells transfected with the empty expression vectors may be used in the same amount as for the SF-TAP-tagged bait protein.However,the tag is quite small and expressed at low levels if not fused to a protein.Thus,the untransfected cell line is an acceptable,simple,and inexpensive alternative for a negative control.Identification of Protein Interactions 19.20.5Current Protocols in Protein Science Supplement 571-4 × 108 HEK293 cell s(1-4 co n fl u e n t 14-cm plate s )expre ss i ng S F-TAP f us io n protei nly s i s(15 mi n 4C)vol u mered u ctio nce n trif ug atio n (10 mi n 10,000 × g )a n aly s i sretai n su per n ata n t fi n alel u atei n c u batio n with50 μl/plate S trep-Tacti n matrix (1 hr)el u tio n with200 μl FLAGel u tio n b u ffer(10 mi n )wa s h 3 time s with 500 μl wa s h b u ffer (s pi n 5 s ec, 100 × g )wa s h 3 time s with500 μl wa s h b u ffer(s pi n 5 s ec, 100 × g )el u tio n with 500 μl de s thiobioti n el u tio n b u ffer (10 mi n )i n c u batio n with25 μl/platea n ti-FLAG M2a g aro s e(1 hr)Figure 19.20.3Flow chart for the S F-T AP proced u re.4.Let cells grow for 48hr.If necessary,cells can be starved in DMEM without FBS for 12hr prior to harvesting.Starving might be desirable if cell signaling is to be analyzed,especially prior to differ-ential treatment with growth factors,to eliminate effects of serum growth factors.Lyse cells5.Remove medium from the plates.6.Optional:Rinse cells in warm PBS.Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.6Supplement 57Current Protocols in Protein Science7.Scrape off cells in 1ml lysis buffer per 14-cm plate on ice using a cell scraper,and combine lysates from each experimental condition in a 1.5-ml microcentrifuge tube.8.Lyse cells by incubating 15min on ice with mixing by hand from time to time.9.Pellet cell debris,including nuclei,by centrifuging 10min at 10,000×g ,4◦C.10.Clear lysate supernatant by filtration through a 0.22-μm syringe filter.Perform SF-TAP 11.Wash Strep-Tactin Superflow resin twice,each time with 4resin volumes TBS and once with 4resin volumes lysis buffer.12.Incubate lysates with 50μl per 14-cm plate of settled Strep-Tactin Superflow resin for 1hr at 4◦C (use an end-over-end rotator to keep the resin evenly distributed).Note that a maximum of 200μl settled resin per spin column should not be exceeded.If more than four 14-cm plates (∼4×108HEK293cells)are used,reduce the volume per plate or use additional spin columns in step 13.13.Centrifuge for 30sec at 7000×g ,4◦C,remove the supernatant until 500μl remains,and transfer resin to a microspin column.Snap off bottom closure of the spin column prior to use.The maximum volume of the spin columns is 650μl.Alternatively,centrifugations for wash and elution steps can be performed at room temperature if no cooled centrifuge is available.14.Remove remaining supernatant by centrifugation in the spin column for 5sec at 100×g ,then wash resin three times,each time with 500μl wash buffer (centrifuge 5sec at 100×g each time to remove the supernatant)at 4◦C.Replug spin columns with inverted bottom closure prior to adding the elution buffer in step 15.IMPORTANT NOTE:Do not allow the resin to run dry.Depending on the bait protein,this markedly reduces the yield.15.Add 500μl desthiobiotin elution buffer and gently mix the resin by hand for 10min on ice.16.Remove the plug of the spin column,transfer the column to a new collection tube,and collect the eluate by centrifuging 10sec at 2000×g ,4◦C.If spin columns were closed by the top screw cap during incubation with elution buffer,the cap needs to be removed prior to centrifugation,to allow the pressure to balance out.17.Wash anti–FLAG M2agarose resin three times,each time with 4resin volumes TBS.Suspend resin in TBS and transfer it to microspin columns,then remove the buffer by centrifuging 5sec at 100×g .25μl settled resin per 14-cm plate will be needed.18.Transfer eluate from step 16corresponding to each 14-cm plate to a microspin column containing 25μl settled anti-FLAG M2agarose prepared as in step 17.19.Plug columns,close columns with top screw caps,and incubate for 1hr at 4◦C (on an end-over-end rotator).20.Wash once with 500μl wash buffer,and then twice,each time with 500μl TBS (centrifuge 5sec at 100×g each time to remove the supernatant)at 4◦C.21.For elution,incubate with 4bead volumes (at least 200μl)FLAG elution buffer for 10min,keeping the columns plugged and gently mixing the resin several times.22.After incubation,remove the plugs and top screws of the spin columns,transfer to new collection tubes,and collect the eluate(s)by centrifugation (10sec at 2000×g ).Identification of Protein Interactions 19.20.7Current Protocols in Protein Science Supplement 5723.Depending on downstream method to be used,either precipitate protein (see SupportProtocol 2)or concentrate the eluate by Microcon YM-3centrifugal filter units according to manufacturer’s protocols.SUPPORT PROTOCOL 1GENERATION OF HEK293CLONES STABLY EXPRESSINGSF-TAP-TAGGED PROTEINSIn Basic Protocol 1,SF-TAP-tagged proteins are transiently expressed.However,strong overexpression of the bait protein usually increases copurification of heat-shock proteins such as HSP70.For in-depth analysis,it is therefore recommended to generate cell lines stably expressing the bait protein.This protocol presents a quick method for generating stable HEK293lines.MaterialsHEK293cells (ATCC no.CRL-1573)Complete DMEM containing 10%FBS (APPENDIX 3C )SF-TAP vectors with appropriate insert,and empty control plasmid (see Critical Parameters)Transfection reagent of choice (see UNIT 5.10)Phosphate-buffered saline (PBS;APPENDIX 2E )Complete DMEM medium (APPENDIX 3C )G418(PAA Laboratories, )Freezing solution:90%fetal bovine serum (FBS;Invitrogen)/10%dimethylsulfoxide (DMSO;AR grade)Lysis buffer (see recipe)Blocking reagent:5%(w/v)nonfat dry milk in TBS (see recipe for TBS)containing 0.1%(v/v)Tween 20Anti-FLAG M2antibody (Sigma-Aldrich)10-cm tissue culture dishes12-well and 6-welll tissue culture platesCentrifuge2-ml cryovials (Nunc)Additional reagents and equipment for transfection of mammalian cells (UNIT 5.10),trypsinization and counting of cells (UNIT 5.10),and immunoblotting (UNIT 10.10)Grow and transfect cells1.Grow cells in complete DMEM containing 10%FBS.2.Transfect cells with expression plasmid using a transfection reagent of choice ac-cording to the manufacturer’s protocols.3.Change medium after 6hr.Select cells4.After 48hr,trypsinize and count cells (APPENDIX 3C )and seed them at low density (1×106cells per 10-cm dish)to allow formation of single colonies upon selection.5.Add G418(500to 1000μg/ml)for selection of the SF-TAP expression vectors,which are based on pcDNA3.0and contain a neomycin-resistance gene.6.Grow the cells under G-418selection for 2to 4weeks,changing the medium every second day.7.Collect single colonies with a 200-μl pipet into 12-well plates.8.Keep colonies under G418selection until the cell density is sufficient for expanding them to 6-well dishes (two wells per clone).Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.8Supplement 57Current Protocols in Protein ScienceCryopreserve cells 9.Grow cells to >90%confluency and trypsinize (APPENDIX 3C )one well of each clone for generation of cryostocks.10.Generate cryostocks:a.Wash cells from one well once by adding 3ml PBS,centrifuging 5min at 800×g ,room temperature,and resuspending the pellet in 500μl freezing buffer.b.Transfer resuspended cells to 2-ml cryovials.c.Freeze cells slowly:keep cells for 1hr at −20◦C,then overnight at −80◦C,followed by storage in a liquid nitrogen tank.For cultivation and expansion of confirmed clones,thaw the cryostock at 37◦C,wash cells once with medium,and plate cells onto 10-cm culture dishes.Test for expression of bait protein 11.Lyse one well of each clone in 300μl lysis buffer and test for expression of the bait protein by immunoblotting (UNIT 10.10).SF-TAP proteins can be detected using the anti-FLAG M2antibody (Sigma-Aldrich)at a dilution of 1:1000to 1:5000in blocking reagent.SUPPORTPROTOCOL 2CHLOROFORM/METHANOL PRECIPITATION OF PROTEINS The chloroform/methanol precipitation method described by Wessel and Fl¨u gge (1984)precipitates proteins with high efficiency and yields samples containing low levels of salt contamination.Materials SF-TAP eluate (from Basic Protocol 1)Methanol (AR grade)Chloroform (AR grade)2-ml polypropylene sample tubes 1.Transfer 200μl SF-TAP eluate to a 2-ml sample tube.All steps are performed at ambient temperature.2.Add 0.8ml of methanol,vortex,and centrifuge for 20sec at 9000×g ,room temperature.3.Add 0.2ml chloroform,vortex,and centrifuge for 20sec at 9000×g ,room temperature.4.Add 0.6ml of deionized water,vortex for 5sec,and centrifuge for 1min at 9000×g ,room temperature.5.Carefully remove and discard the upper layer (aqueous phase).The protein precipitate (visible as white flocks)is in the interphase.6.Add 0.6ml of methanol,vortex,and centrifuge for 2min at 16,000×g ,room temperature.7.Carefully remove the supernatant and air dry the pellet.The pellet can be stored for several months at –80◦C.Identification of Protein Interactions 19.20.9Current Protocols in Protein Science Supplement 57BASIC PROTOCOL 2IN-SOLUTION DIGEST OF PROTEINS FOR MASS SPECTROMETRIC ANALYSISThe in-solution digest described here is a quick and efficient method to digest the SF-TAP eluate after protein precipitation (Support Protocol 2).The use of an MS-compatible surfactant helps to solubilize the precipitated proteins.In order to allow the identification of cysteine-containing peptides,random oxidation is prevented,rather than reverted,by applying a DTT/iodoacetamide treatment prior to digestion,leading to a defined-mass adduct.The digested protein sample can then be directly subjected to analysis on an LC-coupled tandem mass spectrometer.MaterialsPrecipitated protein (see Support Protocol 2)50mM ammonium bicarbonate (freshly prepared)RapiGest SF (Waters):prepare 2%(10×)stock solution in deionized water 100mM DTT (prepare from 500mM stock solution;store stock up to 6months at −20◦C)300mM iodoacetamide (prepare fresh)50×(0.5μg/μl)trypsin stock solution (Promega;store at −20◦C)Concentrated (37%)HCl60◦C incubatorPolypropylene inserts (Supelco,cat.no.24722)1to 200μl gel-loader pipet tips (Sorenson Bioscience,/contact.cfm )1.Dissolve the protein pellet in 30μl of 50mM ammonium bicarbonate by extensive vortexing.2.Add 3μl of 10×(2%)RapiGest stock solution (final concentration,0.2%).RapiGest (sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl]-1-propanesulfo-nate)is an acid-labile surfactant that helps to solubilize and denature proteins to make them accessible to proteolytic digestion (Yu et al.,2003).3.Add 1μl of 100mM DTT and vortex.4.Incubate 10min at 60◦C.5.Cool the samples to room temperature.6.Add 1μl of 300mM iodoacetamide and vortex.7.Incubate for 30min at room temperature.Samples should be protected from light,since iodoacetamide is light-sensitive.8.Add 2μl trypsin stock solution and vortex.9.Incubate at 37◦C overnight.10.Add 2μl of concentrated (37%)HCl to hydrolyze the RapiGest.For hydrolysis of the RapiGest reagent,the pH must be <2.11.Transfer samples to polypropylene inserts (remove spring).12.Incubate for 30min at room temperature.13.Place inserts in 1.5-ml microcentrifuge tubes and microcentrifuge 10min at 13,000×g ,room temperature.One hydrolysis product of the RapiGest reagent is water-immiscible and can be removed by centrifugation.After centrifugation,it is visible as faint film (oleic phase)on top of theStrep/FLAGTandem Affinity Purification (SF-TAP)19.20.10Supplement 57Current Protocols in Protein Science aqueous sample phase.The other hydrolysis product is an ionic water-soluble component which does not interfere with reversed phase LC or MS analysis.A white pellet might appear.14.Carefully recover the solution between the upper oleic phase and the pellet using gel-loader tips.The sample can now be directly subjected to C18HPLC separation prior to MS/MS-analysis (LC-MS/MS;Basic Protocol 4).Pre-fractionation (Basic Protocol 3)is optional.BASIC PROTOCOL 3PRE-FRACTIONATION VIA SDS-PAGE AND IN-GEL DIGESTION PRIOR TO LC-MS/MS ANALYSIS Pre-fractionation prior to MS analysis increases the number of peptides which can be an-alyzed,and therefore the peptide coverage of identified proteins.This benefit is achieved by overcoming the undersampling problem mainly caused by the limited capacity of the trapping columns used in nano–LC chromatography,or that occurs with high complexity.For these samples,SDS-PAGE pre-fractionation can be used to reduce the complexity.For less complex samples or samples with low protein content,the in-solution digest (Basic Protocol 2)is preferred.Materials Protein sample (e.g.,from Basic Protocol 1or Support Protocol 2)10%NuPAGE gels (Invitrogen)MOPS running buffer (Invitrogen)40%and 100%acetonitrile (AR grade;prepare fresh)5mM DTT (prepare from 500mM stock;store stock up to 6months at −20◦C)25mM iodoacetamide (prepare fresh)Digestion solution:dilute 50×trypsin stock solution (0.5μg/μl,Promega)1:50in 50mM ammonium bicarbonate (freshly prepared)1%and 0.5%(v/v)trifluoroacetic acid (TFA;prepare fresh from 10%v/v stock)50%(v/v)acetonitrile/0.5%(v/v)TFA (prepare fresh)99.5%(v/v)acetonitrile/0.5%(v/v)TFA (prepare fresh)2%(v/v)acetonitrile/0.5%(v/v)TFA Concentration units (e.g.,Microcon from Millipore)Scalpel Polypropylene 96-well microtiter plate:polystyrene material should be avoided since,depending on the product,polymers can be extracted from plastics which produce strong background signals in mass spectrometry 60◦C incubator or heating block Polypropylene 0.5-ml reaction tubes Microtiter plate shaker (e.g.,V ortex mixer equipped with microtiter-plate adaptor)HPLC sample tubes Additional reagents and equipment for SDS-PAGE (UNIT 10.1)and colloidal Coomassie blue staining of gels (Support Protocol 3)Prepare samples 1.Concentrate samples using concentration units (e.g.,Microcon).2.Supplement samples with Laemmli loading buffer (SDS-PAGE loading buffer;UNIT 10.1).A detailed description of the SDS gel electrophoresis and standard buffers can be found in UNIT 10.1or in the protocols supplied with the NuPAGE system.Identification of ProteinInteractions19.20.11Perform electrophoresis and stain gels3.Separate samples on 10%NuPAGE gels according to the manufacturer’s protocols,using MOPS running buffer.4.Stop electrophoresis after the gel front has travelled 1to 2cm.5.Stain gels with colloidal Coomassie blue (see Support Protocol 3).Avoid strong staining of the bands since it increases the time necessary for destaining.6.Excise desired gel pieces with a clean scalpel (three to ten slices,depending on the complexity of the sample).Destain and process gel slices7.Transfer gel pieces into individual wells of a 96-well plate.8.Wash by adding 100μl water to each well and incubating for 30min.9.For destaining:a.Wash twice,each time by incubating the gel slices for 10min in 100μl/well of 40%acetonitrile.b.Wash for 5min in 100μl/well of 100%acetonitrile (if gels are still blue,repeat de-staining).10.Add 100μl of 5mM DTT,then incubate 15min at 60◦C in an incubator or heatingblock.11.Remove DTT solution and cool the plate to room temperature.12.Add 100μl per well of freshly prepared 25mM iodoacetamide,then incubate 30minin the dark.13.Wash twice,each time for 10min with 100μl/well of 40%acetonitrile.14.Wash 5min with 100μl/well of 100%acetonitrile.15.Discard supernatant and air dry (or SpeedVac)the gel pieces to complete dryness.Digest and extract gel slices16.Add 20to 30μl per well of freshly prepared digestion solution (depending on the sizeof the gel plugs).Wrap plates in Parafilm to reduce evaporation during the overnight incubation (or use a humidified incubator in step 17).17.Digest overnight at 37◦C.18.For extraction of the peptides from the gel piece,add 10μl 1%TFA,then shake15min on a V ortex mixer with a microtiter plate adapter.The peptides are extracted in three steps with increasing acetonitrile concentrations (steps 18to 23).19.Transfer liquid (extract 1)to a 0.5-ml polypropylene tube.20.Add 50μl 50%acetonitrile/0.5%TFA to the gel piece and shake 15min on a V ortexmixer with a microtiter plate adapter.21.Remove the liquid (extract 2)and pool extracts 1and 2.22.Add 50μl 99.5%acetonitrile/0.5%TFA to the gel piece,then shake 15min on aV ortex mixer with a microtiter plate adapter.23.Remove the liquid (extract 3)and pool extract 3with 1and 2.Strep/FLAG Tandem AffinityPurification(SF-TAP)19.20.1224.Dry samples to complete dryness in a SpeedVac evaporator.25.Redissolve samples in50μl of2%acetonitrile/0.5%TFA by shaking(e.g.,on aV ortex mixer)for10to15min,then transfer the sample into HPLC sample tubes for LC-MS/MS analysis.SUPPORT PROTOCOL3QUICK MS-COMPATIBLE COLLOIDAL COOMASSIE STAIN OF PROTEINS AFTER SDS-PAGE SEPARATIONThe colloidal Coomassie stain(Kang et al.,2002)represents a fast and sensitive MS-compatible protein staining method.In contrast to the classical staining protocol,no intense and time-consuming destaining is needed to visualize protein bands.Therefore, this method is ideal for a quick staining of the protein bands and provides good orientation on how the gel can be fractionated without splitting predominant bands(see Basic Protocol3).MaterialsElectrophoresed SDS gel containing protein samples of interest(e.g.,from Basic Protocol3)Colloidal Coomassie staining solution(see recipe)Destaining solution:10%(v/v)ethanol/2%(v/v)orthophosphoric acidGel staining trays of appropriate size1.Wash gels twice,each time for10min in deionized water in a staining tray.The SDS must be removed before staining to reduce background signals.2.Incubate gels for10min in colloidal Coomassie staining solution.The incubation steps are kept short for the staining of gels used for pre-fractionation.The staining can be prolonged up to overnight.The maximum staining will be reached after ∼3hr incubation in the staining solution.3.Incubate gels for10min in destaining solution.4.Wash gels twice,each time for10min in deionized water.BASIC PROTOCOL4LC-MS/MS ANALYSIS OF DIGESTED SF-TAP SAMPLESThe following protocol describes MS analysis of digested protein samples on an LC-coupled ESI tandem mass spectrometer.The representative MS-analysis protocol is provided for an Orbitrap mass spectrometer(Thermo Fisher Scientific).The Orbitrap system combines fast data acquisition with high mass accuracy and is therefore ideal for the analysis of SF-TAP samples.Background information on mass spectrometric analysis can be found in UNIT16.11.MaterialsDigested protein sample,either from in-solution digest(Basic Protocol2)or in-gel digest(Basic Protocol3)Nano HPLC loading buffer:0.1%formic acid in HPLC-grade waterNano HPLC buffer A:2%acetonitrile/0.1%formic acid in HPLC-grade waterNano HPLC buffer B:80%acetonitrile/0.1%formic acid in HPLC-grade water HPLC vials(Dionex)Nano HPLC system(UltiMate3000,Dionex)equipped with a trap column (100μm i.d.×2cm,packed with Acclaim PepMap100C18resin,5μm,100◦A;Dionex)and an analytical column(75μm i.d.×15cm,packed with AcclaimPepMap100C18resin,3μm,100◦A;Dionex)Mass spectrometer:Oritrap XL with a nanospray ion source(ThermoFisher Scientific;also see UNIT16.11)。
Novagen S
Induction of λDE3 Lysogens After a target plasmid is established in BL21(DE3), HMS174(DE3), NovaBlue(DE3) or in one of these strains containing pLysS or pLysE, expression of the target DNA is induced by the addition of IPTG to a growing culture. For pET constructions carrying the “plain” T7 promoter (e.g. pSCREEN), a final concentration of 0.4mM IPTG is recommended, while 1mM IPTG is recommended with vectors having the T7lac promoter (e.g. pET-29, pET-30 and pET-32). An example of an induction protocol is presented below.
Total Cell Protein
The expression of target genes may be quickly assessed by analysis of total cell protein on an SDS-polyacrylamide gel followed by Coomassie blue staining. Collect induced cells by centrifugation, resuspend in 1/10 culture volume of 10mM Tris-HCl pH 8.0, 2mM EDTA, remove an aliquot and add to it an equal volume of 2X SDS sample buffer. Heat to 70°C for 5 min and load 5-20µl on a gel. The proper amount of material to load depends on the cell density at time of harvest and the expression level of the target protein. Usually, an amount equivalent to 15µl of a culture with an OD600 of 1.5 (3µl of sample by the above method) gives the proper band intensities with Coomassie blue staining. Much less protein (~1/500 of this amount) is required for Western blot or dot blot analysis with the S-protein HRP Conjugate.
国家人类基因组北方研究中心
Functional Proteomics – To study protein-protein interaction, 3-D structures, cellular localization and PTMs in order to understand the physiological function of the whole set of proteome.
Anal Biochem 1991, 199:223-231
Blue Native PAGE
_ detergent
CBB
6-ACA
+
Blue Native PAGE
Sample Preparation Solubilization with nonionic detergent (laurylmaltoside, TX-100, CHAPS, Mega 9, octylglucoside, Brij 35, etc), supplemented with 6-aminocaproic acid Separation gel: 6-13% gradient Cathode buffer contains 0.02% Coomassie blue G250 Separation of members of multiprotein complex
Blue Native PAGE
Seeing the Unseeable The Science of Cellular Simulation - Human 看了看不见的细胞模拟人类的科学
Differential Eqs
How To Do it? (Computationally)
Boolean Networks
Reservoir Analysis
Electrical Circuit Model
Cellular Automata
Who’s Doing It?
• E-cell Project (Keio University, Japan) • BioSpice Project (Arkin, Berkeley) • Metabolic Engineering Working Group
Cellular Automata
• Computer modelling method that uses lattices and discrete state “rules” to model time dependent processes – a way to animate things
• Does not reproduce physical events (brownian motion, collisions, transport, binding, etc.) that might be seen in a cell – Petri Nets are more like a plumbing and valving control system What about Cellular Automata?
• Differential equations are “hard” to work with when simulating spatial phenomena, when dealing with discrete events (binding, switching), non continuous variables (low copy number) or when key parameters are unknown or unknowable
蛋白质组学介绍
• Acetylation - the addition of an acetyl group, usually at the N-terminus of the protein
• Alkylation - the addition of an alkyl group (e.g. methyl, ethyl)
• A new fundamental concept called proteome (PROTEin complement to a genOME) has recently emerged.
Proteomics
• should drastically help to unravel biochemical and physiological mechanisms of complex multivariate diseases at the functional molecular level.
• However, the understanding of probably half a million human proteins encoded by less than 30,000 genes is still a long way away and the hard work to unravel the complexity of biological systems is yet to come.
be non-functional.
• D -Exon cassette mode: Certain exons are spliced out to alter the sequence of amino acids in the expressed protein.
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arXiv:q-bio/0312033v2 [q-bio.BM] 6 Feb 2004Specificprotein-proteinbindinginmany-componentmixturesofproteins
RichardP.SearDepartmentofPhysics,UniversityofSurrey,Guildford,SurreyGU27XH,UnitedKingdomr.sear@surrey.ac.uk
Abstract.Proteinsmustbindtospecificotherproteinsinvivoinordertofunction.Theproteinsmustbindonlytooneorafewotherproteinsoftheoforderathousandproteinstypicallypresentinvivo.Usingasimplemodelofaprotein,specificbindinginmanycomponentmixturesisstudied.Itisfoundtobeademandingfunctioninthesensethatitdemandsthatthebindingsitesoftheproteinsbeencodedbylongsequencesofelements,andtherequirementforspecificbindingthenstronglyconstrainsthesesequences.Thisisquantifiedbythecapabilityofproteinsofagivensize(sequencelength),whichisthemaximumnumberofspecific-bindinginteractionspossibleinamixture.ThiscalculationofthemaximumnumberpossibleisinthesamespiritastheworkofShannonandothersonthemaximumrateofcommunicationthroughnoisychannels.Specificprotein-proteinbindinginmany-componentmixturesofproteins21.IntroductionProteinsmustinteracttofunction,andtheymustinteractwithveryhighspecificityinthehighlycomplexmixturesthatlieinsidecells.Forexample,the3proteinsCdk2,cyclinandPCNAbindtogetherinthenucleustoformacomplex,whichthengoesontointeractwithhighspecificitywithafewotherproteins[1].(PCNA=ProliferatingCellNuclearAntigenandCdk2=Cyclindependentkinase;theyarebothinvolvedincelldivisionbutalsoinotherprocesses.)Cdk2mustbindstronglytocyclinandtoPCNA,i.e.,thecomplexmusthavealargeequilibriumconstantorequivalentlyasmalldissociationconstant.ButCdk2mustnotbindtotheoforderonethousandotherproteinspresenti.e.,theequilibriumconstantswithalltheseotherproteinsmustbesmall.Thishighlyspecificandstrongbindinginamixtureofthousandsofdifferentmoleculesisademandingrequirementandherewewilltrytoquantifyhowdemandingitisforaverysimplemodelofaprotein.Todoso,wewillrelyonthefactthatspecificbindinginamixtureisanalogoustocommunicationthroughanoisychannel,whichhasbeenextensivelystudied,inparticularbyShannon[2,3]andthemanywhohavebuiltonhiswork.Incommunicationthroughanoisychannelreliablecommunicationrequiresthatallpossiblemessagesbesufficientlydistinctfromeachotherevenafterdistortionbythenoise.Inspecificbindinginamixture,aproteinbindingsiteorpatchonthesurfaceofaproteinmustbesufficientlydistinctfromallotherpatchestoallowoneotherpatchtobindtoitwithanequilibriumconstantmuchlargerthanthatofanyotherinteractionofthepatch.ThetheorydevelopedbyShannonhasbeenenormouslyusefulinunderstandingcommunicationsystemsandbuildingefficientones.Wehopethatapplyingthetheorytoprotein-proteininteractionsincellswillbesimilarlyuseful.ThisanalogytocommunicationhasbeenusedforthesimilarproblemofbindingbetweenaproteinandaspecificDNAsequence,inparticularbySchneider[4–7].Seealso[8]forotherrelatedwork.Similarapproacheshavealsobeenusedextensively,seethereview[9],instudiesofourimmunesystem,see[10]foranintroductiontoourimmunesystem.Studiesoftheimmunesystemlookatalargesetofimmune-systemproteinsrecognisingasingleforeignprotein.Bycontrast,oursituation,amodelofthecytoplasmofaprokaryoteoracompartmentofaeukaryote,involvesnotasingleproteinbindingtoanotherbutbindingbetweenaverylargenumberofpairsofproteins.However,therearecloseanalogiesbetweenthespecificbindingrequiredinsideallcellsandthatrequiredofimmune-systemproteins,inparticularinbothcasesproteinsmustavoidbindingtothewrongprotein[9,11,12].Insidecells,thiswouldbeallproteinsbaraprotein’spartner,whereasinthecaseoftheimmunesystem,itsproteinsmustnotbindtootherhumanproteins.OurexampleofCdk2needingtobindtoPCNAandtocyclin,istheruleratherthantheexceptionineukaryotecells[10].Wemerelychoseaspecificexampletomakethediscussionmoreconcrete.Thecellsrelytotallyonacomplexnetworkorwebofmanyspecificinteractions.Thislargesetofinteractionsisoftencalledtheinteractome,byanalogytotheuseofthewordgenometodenotethesetofgenesofanorganism.Inthestudyofnoisychannels,afundamentalquantityofinterestisthechannelcapacity[2,3,13]:themaximumnumberofbitsofinformationthatcanbesentpersecond.Shannonderivedhisfamouschannel-capacitytheorem[2,3]whichallowsthischannelcapacitytobecalculated.Herewewillcalculatearelatedquantityforproteins,whichwewillcallthecapability.Itisthemaximumnumberof