Psalmotoxin-1(PcTx1,Pi-theraphotoxin-Pc1a) hasbeenisolatedfromthevenomoftheSpiderPsalmopoeuscambridgei(Trinidadchevrontarantula). PcTx1 isknowntoblockpotently(IC50 =1nM)andselectivelythe H+-gatedsodiumchannelASIC1a (acid-sensitiveionchannel1a).TheblockageisrapidandreversIBLe. PcTx1 candistinguishbetweenthetwoASIC1splicevariantsASIC1aandASIC1b. PcTx1 losesitscapacitytoblockASIC1aassoonasthissubunitisassociatedwithanothermemberofthefamily(ASIC2aorASIC3).PcTx1demonstratesananalgesiceffectinacuteandneuropathicpainmodels.
Fig1:effectof30nMPcTx1#13PCT001onASIC1aexpressedinoocytes. Inhibitionisnearlycompleteatthisconcentration.
Description:
AAsequence: Glu-Asp-Cys3-Ile-Pro-Lys-Trp-Lys-Gly-Cys10-Val-Asn-Arg-His-Gly-Asp-Cys17-Cys18-Glu-Gly-Leu-Glu-Cys23-Trp-Lys-Arg-Arg-Arg-Ser-Phe-Glu-Val-Cys33-Val-Pro-Lys-Thr-Pro-Lys-Thr-OH
Disulfidebonds: Cys3-Cys18,Cys10-Cys23 andCys17-Cys33
Length(aa): 40
Formula: C200H312N62O57S6
MolecularWeight: 4690.82Da
Appearance:Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: notavailable
Source: Synthetic
Purityrate: >98%
Reference:
HeteromericAcid-SensingIonChannels(ASICs)ComposedofASIC2bandASIC1aDisplayNovelChannelPropertiesandContributetoAcidosis-InducedNeuronalDeath
Acid-sensingionchannel(ASIC)subunitsassociatetoformhomomericorheteromericproton-gatedionchannelsinneuronsthroughoutthenervoussystem.TheASIC1asubunitplaysanimportantroleinestablishingthekineticsofproton-gatedcurrentsintheCNS,andactivationofASIC1ahomomericchannelsinducesneuronaldeathafterlocalacidosisthataccompaniescerebralischemia.TheASIC2bsubunitisexpressedinthebraininapatternthatoverlapsASIC1a,yetthecontributionofASIC2bhasremainedelusive.WefindthatcoexpressionofASIC2bwithASIC1ainXenopusoocytesresultsinnovelproton-gatedcurrentswithpropertiesdistinctfromASIC1ahomomericchannels.Inparticular,ASIC2b/1aheteromericchannelsareinhibitedbythenonselectivepotassiumchannelblockerstetraethylammoniumandbarium.Inaddition,steady-statedesensitizationisinducedatmorebasicpHvalues,andBigDynorphinsensitivityisenhancedintheseuniqueheteromericchannels.Culturedhippocampalneuronsshowproton-gatedcurrentsconsistentwithASIC2bcontribution,andthesecurrentsarelackinginneuronsfrommicewithanACCN1(ASIC2)genedisruption.Finally,wefindthattheseASIC2b/1aheteromericchannelscontributetoacidosis-inducedneuronaldeath.Together,ourresultsshowthatASIC2bconfersuniquepropertiestoheteromericchannelsincentralneurons.FurThermore,thesedataindicatethatASIC2,likeASIC1,playsaroleinacidosis-inducedneuronaldeathandimplicatetheASIC2b/1asubtypeasanovelpharmacologicaltargettopreventneuronalinjuryafterstroke.
SherwoodTW.,etal.(2011)HeteromericAcid-SensingIonChannels(ASICs)ComposedofASIC2bandASIC1aDisplayNovelChannelPropertiesandContributetoAcidosis-InducedNeuronalDeath.TheJournalofNeuroscience. PMID: 21715637
Psalmotoxin-1dockingtohumanacid-sensingionchannel-1
Acid-sensingionchannel-1(ASIC-1)isaproton-gatedionchannelimplicatedinnociceptionandneuronaldeathduringischemia.RecentlythefirstcrystalstructureofachickenASICwasobtained.Expandinguponthiswork,homologymodelsofthehumanASICswereconstructedandevaluated.Energy-minimizedstructuresweretestedforvaliditybyinsilicodockingofthemodelstopsalmotoxin-1,whichpotentlyinhibitsASIC-1andnotothermembersofthefamily.ThedataareconsistentwithpriorrADIoligandbindingandfunctionalassayswhilealsoexplainingtheselectivityofPcTX-1forhomomerichASIC-1a.Bindingenergycalculationssuggestthatthetoxinandchannelcreateacomplexthatismorestablethanthechannelalone.Thebindingisdominatedbythecoulombiccontributions,whichaccountforwhythetoxin-channelinteractionisnotobservedatlowpH.Thecomputationaldatawereexperimentallyverifiedwithsinglechannelandwhole-cellelectrophysiologicalstudies.Thesevalidatedmodelsshouldallowfortherationaldesignofspecificandpotentpeptidomimeticcompoundsthatmaybeusefulforthetreatmentofpainorischemicstroke.
QadriYJ., etal. (2009)Psalmotoxin-1dockingtohumanacid-sensingionchannel-1. JBC. PMID: 19395383
AtarantulapeptideagainstpainviaASIC1achannelsandopioidmechanisms
Psalmotoxin1,apeptideextractedfromtheSouthAmericantarantulaPsalmopoeuscambridgei,hasverypotentanalgesicpropertiesagainstthermal,mechanical,chemical,inflammatoryandneuropathicpaininrodents.Itexertsitsactionbyblockingacid-sensingionchannel1a,andthisblockaderesultsinanactivationoftheendogenousenkephalinpathway.TheanalgesicpropertiesofthepeptidearesuppressedbyantagoNISTsofthemuanddelta-opioidreceptorsandarelostinPenk1-/-mice.
MazzucaM., etal. (2007)AtarantulapeptideagainstpainviaASIC1achannelsandopioidmechanisms. NatNeurosci.PMID: 17632507
CationselectivityandinhibitionofmalignantgliomaNa+channelsbyPsalmotoxin1
Psalmotoxin1(acomponentofthevenomofaWestIndiestarantula)isa40-aminoacidpeptidethatinhibitscationcurrentsmediatedbyacid-sensingionchannels(ASIC).InthisstudyweperformedelectrophysiologicalexperimentstotestthehypothesisthatPsalmotoxin1(PcTX1)inhibitsNa+currentsinhigh-gradehumanastrocytomacells(glioblastomamultiforme,orGBM).Inwholecellpatch-clampedculturedGBMcells,thepeptidetoxinquicklyandreversiblyinhibitedbothinwardandoutwardcurrentwithanIC50of36+/-2pM.ThesameinhibitionwasobservedinfreshlyresectedGBMcells.However,whenthesameexperimentwasperformedonnormalhumanastrocytes,thetoxinfailedtoinhibitthewholecellcurrent.WealsodeterminedacationicselectivitysequenceforinwardcurrentsinthreeculturedGBMcelllines(SK-MG-1,U87-MG,andU251-MG).TheselectivitysequenceyieldedauniquebiophysicalfingerprintwithinwardK+conductanceapproximatelyfourfoldgreaterthanthatofNa+,Li+,andCa2+.TheseobservationssuggestthatPcTX1mayproveusefulindeterminingwhetherGBMcellsexpressaspecificASIC-containingionchanneltypethatcanserveasatargetforbothdiagnosticandtherapeutictreatmentsofaggressivemalignantgliomas.
BubienJK., etal. (2004)CationselectivityandinhibitionofmalignantgliomaNa+channelsbyPsalmotoxin1. AmJPhysiolCellPhysiol. PMID: 15253892
RecombinantproductionandsolutionstructureofPcTx1,thespecificpeptideinhibitorofASIC1aproton-gatedcationchannels
Acid-sensingionchannels(ASICs)arethoughttobeimportantionchannels,particularlyfortheperceptionofpain.Someofthemmayalsocontributetosynapticplasticity,learning,andmemory.Psalmotoxin1(PcTx1),thefirstpotentandspecificblockeroftheASIC1aproton-sensingchannel,hasbeensuccessfullyexpressedintheDrosophilamelanogasterS2cellrecombinantexpressionsystemusedhereforthefirsttimetoproduceaspidertoxin.Therecombinanttoxinwasidenticalinallrespectstothenativepeptide,anditsthree-dimensionalstructureinsolutionwasdeterminedbymeansof(1)H2DNMRspectroscopy.SurfacecharacteristicsofPcTx1provideinsightsonkeystructuralelementsinvolvedinthebindingofPcTx1toASIC1achannels.Theyappeartobelocalizedinthebeta-sheetandthebeta-turnlinkingthestrands,asindicatedbyelectrostaticanisotropycalculations,surfacechargedistribution,andthepresenceofresiduesknowntobeimplicatedinchannelrecognitionbyotherinhibitorcystineknot(ICK)toxins.
Darbon,H., etal. (2003)RecombinantproductionandsolutionstructureofPcTx1,thespecificpeptideinhibitorofASIC1aproton-gatedcationchannels, ProteinSci. PMID: 12824480
Isolationofatarantulatoxinspecificforaclassofproton-gatedNa+channels
Acidsensingisassociatedwithnociception,tastetransduction,andperceptionofextracellularpHfluctuationsinthebrain.Acidsensingiscarriedoutbythesimplestclassofligand-gatedchannels,thefamilyofH(+)-gatedNa(+)channels.Thesechannelshaverecentlybeenclonedandbelongtotheacid-sensitiveionchannel(ASIC)family.Toxinsfromanimalvenomshavebeenessentialforstudiesofvoltage-sensitiveandligand-gatedionchannels.Thispaperdescribesanovel40-aminoacidtoxinfromtarantulavenom,whichpotentlyblocks(IC(50)=0.9nm)aparticularsubclassofASICchannelsthatarehighlyexpressedinbothcentralnervoussystemneuronsandsensoryneuronsfromdorsalrootganglia.ThischanneltypehaspropertiesidenticaltothosedescribedforthehomomultimericassemblyofASIC1a.HomomultimericassembliesofothermembersoftheASICfamilyandheteromultimericassembliesofASIC1awithotherASICsubunitsareinsensitivetothetoxin.Thenewtoxinisthefirsthighaffinityandhighlyselectivepharmacologicalagentforthisnovelclassofionicchannels.Itwillbeimportantforfuturestudiesoftheirphysiologicalandphysio-pathologicalroles.
Lazdunski,M., etal. (2000)Isolationofatarantulatoxinspecificforaclassofproton-gatedNa+channels, JBiolChem. PMID: 10829030
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两个CEX方法A和B测定同一单抗，结果碱性峰比例差不多，酸性峰比例相差约7%，相应主峰也差了7%左右。
具体来说，A方法酸性峰高，主峰低，碱性峰稍微低点；B方法酸性峰低，主峰高，碱性峰稍微高点；另外也做了CIEF，结果呢和A方法更接近。
仔细比较起来，AB两个方法的峰性和数量差不多，就不知道为什么会有这么大的差异。两个方法一个用的WCX柱-磷酸缓冲液，一个用SCX柱-MES缓冲液
大家帮我分析下：
1.两个方法哪个方法更准确，是以酸性峰高的为准还是什么？为什么？
2.这显著差异是由方法造成，具体原因是什么？柱子？
3.CIEF的结果和A方法更接近，是不是可以由此证明A方法更好或者CIEF的方法更好（因为CIEF更快更方便）？
欢迎讨论~
纠正下，A方法用的是Tosoh的柱子，B方法用的是SCX柱。TOSOH的柱子是7um的填料，10cm长。SCX是10um的填料。我本人TOSOH的阳离子柱子用的很少，这次信手用用，结果发现差异很大
那我现在就考虑，在以后方法开发过程中，除了通过流动相pH和组成、梯度、柱子选择来获得样品主峰和酸碱性的最大分离，还要关注各峰比例。因为之前比较方法好坏都只看分离度，尤其是主峰和邻近峰的分离度，获得最大分离度，自然可以做到主峰尽可能纯，但从未认真比较过各峰比例。这是一个大疏忽吧！
另外，CIEF和CEX方法原理还是有点差异的，所以分的是不同的异质体，原液放行两个方法肯定是都要做的。问题就是在早期细胞株筛选和工艺开发阶段，哪个方法才是又快又准。CIEF（iCE280)一般15分钟一个样，比CEX快多了。如果CIEF测得主峰要低于CEX结果，是不是真的完全可以取代CEX呢？CEX分离出的峰远比CIEF的多！
欢迎大家继续讨论~
是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
有了源数据之后把源数据按照大小排列，
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
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