AntibodiescanbeusedinamethodcalledWesternblotting,whichisusefulfordetermininglevelsofproteinexpressionandforassayingproteinsduringpurification.Thismethodusuallyinvolvesthefollowingsteps:

1. Aproteinsampleissubjectedtopolyacrylamidegelelectrophoresis.
2. Afterthisthegelisplacedoverasheetofnitrocelluloseandtheproteininthegeliselectrophoreticallytransferedtothenitrocellulose.
3. Thenitrocelluloseisthensoakedingelatinto"block"itsABIlitytonon-specificallybindproteins.
4. Thenitrocelluloseisthenincubatedwiththespecificantibodyfortheproteinofinterest.
5. Thenitrocelluloseisthenincubatedwithasecondantibodywhichisspecificforthefirstantibody.Forexample,ifthefirstantibodywasraisedinrabbits,thesecondantibodymightbetermed"goatanti-rabbitimmunoglobulin".Whatthismeansisthatrabbitimmunoglobulinswereusedtoelicitanantibodyresponseingoats.Thegoatantibodies(polyclonal)willincludethosewhichrecognizetheconservedregionintherabbitantibodies.SincetheFcregionisconserved,itwillbindtoanyandallrabbitantibodies,includingthoseonthenitrocellulosepaper.
6. Thesecondantibodywilltypicallyhaveacovalentlyattachedenzymewhich,whenprovidedwithachromogenicsubstrate,willcauseacolorreaction.
7. Thusthemolecularweightandamountofthedesiredproteincanbecharacterizedfromacomplexmixture(e.g.crudecellextract)ofotherproteins.

Inavariationoftheabove,theproteinsamplemaybeblotteddirectlyonanitrocellulosepaper(calledadotblot)withoutfirstrunningagel.Thismaybedesirableif,forexample,theantibodyismonoclonalandrecognizesanepitopewhichisdependentuponnativestructure(whichwouldbedestroyeduponrunninganSDSPAGE).

Inadditiontotheirvarieduses,antibodiescanalsobeusedtopurifyproteins.

• Ifrelativelylargeamountsofanantibodycanbeobtained,theycanbecovalentlyattachedtoachromatographyresin(e.g.sephadexbeads).
• Ifacrudecellextractisrunoversuchacolumn,onlytheproteinofinterestshouldbind,andeverythingelsewillflowthrough.
• Theboundproteincanthenbeeluted.ThisistypicallyachievedbymoderatelylowpHconditions(usingaceticacid).AslongastheproteinofinterestisnotirreversIBLydenaturedbysuchconditions,themethodwillworkquitewell.
• Onepotentialpitfallinvolvesthatofmonoclonalantibodiesbeingutilizedtopurifymutantproteins.Theregionsoftheproteincomprisingtheepitopecannotbemodifiedwithoutdestroyingtheabilityoftheantibodytobind.Thus,theuseofmonoclonalantibodiesinapurificationschememayprecludeitsuseinpurifyingcertainmutants.

---

Proteinpurificationcanbethoughtofasaseriesoffractionationstepsdesignedsothat:

• Theproteinofinterestisfoundalmostexclusivelyinonefraction(andwithgoodyield)
• Asignificantamountofthecontaminantscanbefoundinadifferentfraction

Duringpurificationyouwillneedtomonitorseveralparameters,including:

1. Totalsamplevolume
2. Totalsampleprotein
(canbeestimatedbyA₂₈₀;1.4~1.0mg/ml)
3. Unitsofactivityofdesiredprotein
(basedonspecificassay)

Thisbasicinformationwillallowyoutokeeptrackofthefollowinginformationduringeachstepofpurification:

1. %yield
foreachpurificationstep
2. Specificactivity
ofthedesiredprotein(units/mgtotalprotein)
3. Purificationenhancement
ofeachstep(e.g."3.5xpurification)

Indesigningapurificationschemeyoutypicallyhavetobalancepurificationwithyield.

• Forexample,itmayberelativelystraightforwardtoobtain90%purematerialwithgoodyield.
• However,itmaybedifficulttoimprovethatpurityanadditionalfewpercentilewithgoodyield.
• Theplannedapplicationofthepurifiedproteindeterminesthetargetpurity
.
• Iftheproteinistobeusedtodetermineaminoacidsequenceinformation,maybe90%isacceptable.However,ifthematerialistobeusedinclinicaltrials,99.99+%maybethetargetpurity.

Initialstepsinpurification

• Itisextremelyhelpfultohavesomeinformationnotonlyonthegeneralphysicalandchemicalcharacteristicsoftheproteinyouaretryingtopurify,butalsoonthecontaminatingcomponents.
• Forexample,manyE.coliproteinsaregenerallylowmolecularweight(<50,000Da)andsomewhatacidicinisoelectricpoint

Usuallytheinitialstepsinpurificationmakeuseofgeneralphysicaland/orchemicaldifferencesbetweensolubleproteinsandothercellcomponents.

• Forexample,solubleproteinscanbeseparatedfromgeneralcellulardebris,andintactcells,bycentrifugation.
• Thus,cellsarephysicallydisrupted(viahomogenizationoracellpress)toallowreleaseofcellcontents.Thisisthenfollowedbycentrifugationtoseparategenerallysolublecomponentsfromthosewhichareinsoluble.
• Itisatthispointthatdatacollectionbeginsinordertomonitorthepurification.

NucleicacidscansometimesbereADIlyremovedfromthesamplebytheadditionoflargecationiccompoundssuchaspolyethyleneimine,orstreptomycinsulfate.

• Thenucleicacidsbindtothesecompoundsviaelectrostaticinteractionsandthecomplexprecipitatesandcanberemovedviacentrifugation.
• Thesamegeneralresultcanbeobtainedbymixinginionexchangeresinswhichareanionexchangers(i.e.theresinscontaincationicgroups)andthenfilteringorcentrifugingtoremove.Aswitheithermethod,itshouldbeconfirmedthatthedesiredproteinisnotboundaswell.

Crudefractionationsofproteinscanbeachievedbyaddingvariousquantititesofprecipitantssuchasammoniumsulfate,orpolyethyleneglycol(PEG).

• Forthistypeofpurificationstepaninitialexperimentisperformedtomonitorthefractionofoverallprotein,aswellasdesiredprotein,remaininginsolution(andpellet)asafunctionofprecipitantconcentration.

AmmoniumSulfate(%saturated)	0	10	20	30	40	50	60	70	80	90
SampleA280	1000	900	600	200	100	75	50	40	25	20
Activityassay(units)	200	200	200	190	170	100	30	5	0	0 Inthisparticularexampleweareinluck:ataround30%ammoniumsulfatewecanprecipitateabout80%ofthetotalproteinconcentrationinoursample,yetouractivityassayforourdesiredproteinindicatesthatabout95%ofourdesiredproteinisstillsoluble. At80%ammoniumsulfateallofourdesiredproteinhasprecipitated.Thus,fromtheseresultswewoulddothefollowing: Addammoniumsulfatetooursampletoaconcentrationof30%saturation Centrifugeanddiscardthepellet Addammoniumsulfateto80%saturation Centrifugeandkeepthepellet.ResUSPendthepelletinbuffertosolubilizetheprotein. Wewouldexpectabouta5-foldpurificationwithabout95%yield. ================  蚂蚁淘在线  ================ 免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容 版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。 相关阅读 虹膜睫状体免疫细胞活性变化及与角膜移植免... hive官网函数说明文档(原文)_hel... 细解治疗用反义寡核苷酸药物(Therap... KOREAUNITEDPHARMINC_... 首款肺癌全自动免疫组化伴随诊断试剂盒在华... 北京市自来水集团 已有的常见免疫组化难题集锦 诊断试剂 机器学习服务 (Python、R) S... 常见肿瘤免疫标记 IHC免疫组化_组织芯片技术_免疫荧光技... 基于 ENDF/BVII.1 库的 AH... 资质认证 获得国家资质，权威认证！ 全国联保 全国联保，官方无忧售后 正规发票 正规发票，放心购买 签订合同 签订合同，保障您的权益 购物指南 购物流程 注册登录 账户管理 订单查看 常见问题 FAQS 蚁豆兑换 物流说明 评论规则 发票说明 价格说明 支付说明 服务协议 版权声明 诚信保障 免责申明 销售协议 隐私条款 法律申明 售后服务 退换货政策 工作时间 退款说明 售后支持 售后申请表模板列表 关于我们 公司简介 联系我们 资质证书 人才招聘 商务合作 万众创业 网址导航 蚂蚁淘商城 科研狗 苏州蚂蚁淘 生物信息网 现货促销 微信公众号 copyright©2005-2021 蚂蚁淘在线版权所有　苏州蚂蚁淘生物科技有限公司 www.assay-online.com ICP号：苏ICP备17049038号-16 /**/