PlasmidInfo:
PlasmidInformation
ProductName:pSF-OXB20-NH2-6His-MBP-TEV
ProductCode:OG2906
Size(bp):4997bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:OXB20strongconstitutivebacterialpromoter
PlasmidPurpose:
ThisplasmidisdesignedtoexpresstaggedproteinsinE.coli.Theplasmidcontainsaconstitutivepromoter(OXB20)derivedfromtheregionupstreamoftheE.coliRecAgene.Itdoesnotrequireinductionoranyadditionalcomponentsforactivity.Itisthestrongestofthebacterialpromotersthatweprovideandthishighlevelofexpressioncancauseexpressionproblemswithsomeproteinswithpoorsolubility.Forthisreasonwesellarangeofbacterialpromoterswithdifferentexpressionlevels(OXB1(low)>OXB20(high))thatcanbeprovidedwiththepeptidetagsinthisplasmidonrequest.
AbouttheCleavageTag:Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.ThisplasmidcontainsaTEVcleavagetag.Theproteinsequenceofthecleavagetagis:ENLYFQG.CleavageoccursbetweentheGluandGlyresidues.TEVisoftenreportedtohavebetterspecificityforitsrecognitionsitecomparedtoEKTThrombinorFaxtorXa.
Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.
PromoterExpressionLevel:Thisplasmidcontainsaconstitutivebacterialpromoterthatdoesnotrequireinduction.Itisthestrongestbacterialpromoterwesellandthiscancausesolubilityandexpressionproblemswithsomeproteins.WealsoofferarangeofotherbacterialpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
AboutthePeptideTag:Thisplasmidcontainsann-terminalMaltoseBindingProtein(MBP)epitopetagthatcanbefusedtoageneofinteresttoallowproteindetectionand/orpurification.Thesequenceofthetagis:
EEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSS
Formoreinformationonthemethodsthatcanbeusedtopurifyproteinspleaseseeourproteintagguide.
ThisplasmidalsocontainsasecondaryHexa-Histidine(6His)proteintag.Thesequenceofthistagis:HHHHHH
Weprovidearangeofdualpeptidetagplasmids.ThisisbecausesomepeptidetagsprovidespecificBIOLOGicalproperties(e.gsmallmoleculeaffinitynewepitopessolubilityorproteinsecretion)thatarenotprovidedbyothers.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
MakingProteinFusions:ThisplasmidhasbeendesignedtoallowthreetypesofcloningintothemainMCStojoinacodingsequencewiththetag.
1:SnapFusionCloning:IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.
Toinsertyourgene:
1:Amplifyyourgenewithprimersdesignedusingthisspreadsheet
2:CuttheplasmidwitheitherBseRIorBsgI.*
3:Cutyourgenewiththeenzymeyouaddedusingthespreadsheet(anyofAcuIBpmIBpuEIBseRIBsgIEciI).
4:ClonethegeneintotheplasmidusingDNAligase.
UsingthismethodwithanN-terminaltagplasmidwillresultinthetagcodingsequenceimmediatelyfollowedbyyourgenesATGstartcodonatthejoin.Thisresultsinaseamlessfusionofthetwosequenceswithnoextrabasesbeingadded.UsingthismethodonC-terminaltagplasmidswillconvertyourgenesstopcodonintoaTAC(TyrY)codonfollowedbytheplasmidtagcodingsequence.Thisresultsinnoextrabasesbetweenyourgeneandthetag.Seethediagrambelowformoreinformation.
*PleasenotethatinsectexpressionplasmidscannotbecutwithBsgIonlyBseRIbecauseofunavoidableconflictingsitesinthebackbone.AlsoYeastplasmidscannotbecutwithBseRIbecauseofunavoidablerestrictionsitesinthebackbone.
Usingthistechniquewillcreateagenefragmentthatcanbeligatedintoanyorour>1500peptideandreportertagplasmids.Ifyouuseoneoftheothertechniquesbelow(GibsonInFusionSeamlessorLIC)youwillneednewprimersforeveryvectoryoucloneintobecausethearmsofhomologywillchangeaccordingtothetagplasmidyouarecloninginto.
Ifyoufindthatyourgenesequencehassitesinitthatmakeusingthiscloningstrategydifficultyoucanstilluseoneofthealternativemethodsbelow(e.g.standardcloningorGibsoncloning).
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneinourSnapFusiontechnique.
2:StandardEnzymes:Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.
OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.
3:Gibsoncloning/InfusionHD/GeneArtSeamless/LigaseIndependentCloning(LIC)Methods:ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
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1.包被:用0.05MPH9.牰碳酸盐包被缓冲液将抗体稀释至蛋白质含量为1~10μg/ml。在每个聚苯乙烯板的反应孔中加0.1ml,4℃ 过夜。次日,弃去孔内溶液,用洗涤缓冲液洗3次,每次3分钟。(简称洗涤,下同)。
2. 加样:加一定稀释的待检样品0.1ml于上述已包被之反应孔中,置37℃ 孵育1小时。然后洗涤。(同时做空白孔,阴性对照孔及阳性对照孔)。
3. 加酶标抗体:于各反应孔中,加入新鲜稀释的酶标抗体(经滴定后的稀释度)0.1ml。37℃ 孵育0.5~1小时,洗涤。
4. 加底物液显色:于各反应孔中加入临时配制的TMB底物溶液0.1ml,37℃ 10~30分钟。
5. 终止反应:于各反应孔中加入2M硫酸0.05ml。
6. 结果判定:可于白色背景上,直接用肉眼观察结果:反应孔内颜色越深,阳性程度越强,阴性反应为无色或极浅,依据所呈颜色的深浅,以“+”、“-”号表示。也可测OD值:在ELISA检测仪上,于450nm(若以ABTS显色,则410nm)处,以空白对照孔调零后测各孔OD值,若大于规定的阴性对照OD值的2.1倍,即为阳性。
间接法
1.用包被缓冲液将已知抗原稀释至1~10μg/ml,每孔加0.1ml,4℃过夜;
2.次日洗涤3次;
3.加一定稀释的待检样品(未知抗体)0.1ml于上述已包被之反应孔中,置37℃孵育1小时,洗涤;
4.(同时做空白、阴性及阳性孔对照)于反应孔中,加入新鲜稀释的酶标第二抗体(抗抗体)0.1ml;
5.37℃孵育35-60分钟,洗涤;
6.’最后一遍用DDW洗涤。
其余步骤同“双抗体夹心法”的4、5、6。
目前,我用该蛋白的鼠源性单抗来包被ELISA板子,5%BSA37度封闭二小时后,洗涤四次后将板子分三个组加入抗原:其中一个组中加入的是我重组的蛋白,另一个组为用其它方法证实含该蛋白的人体内血清,最后一个组加入PBS。37度一小时后洗涤四次后每孔加入我制备的兔源性多抗。37度一小时洗涤六次后我再加入酶标记的抗兔的抗体37度40分钟后洗涤六次后TMB显色。
问题就在我显色后所有的孔都是阳性,其中以抗原为我的重组蛋白组与PBS组OD值最高,两组都可以达到1.8以上,而加入的抗原为人体血清组低,为0.6-1.0不等。
后面我优化条件,封闭液用过5%的脱脂奶粉,2%的BSA,包被的单抗浓度摸了梯度,加入的多抗也摸了梯度,酶标的抗体也摸了梯度。结果都没有改变,连趋势每次都是一样以重组蛋白组与PBS组OD最高。
今天我还试着把抗原换成其它蛋白也做出了强阳性?
请教各位高手,我的ELISA该如何往下面做了啊,是不是我的抗体本身有问题呢?
对了,我包被用的单抗是用GE公司的预装柱纯化的,很纯,在考染中是看不到一条杂带的。
1. 包被:用0.05M PH9,碳酸盐包被缓冲液将抗体稀释至蛋白质含量为1~10μg/ml,在每个聚苯乙烯板的反应孔中加0.1ml,4℃过夜。次日,弃去孔内溶液,用洗涤缓冲液洗3次,每次3分钟。
2. 加样:加一定稀释的待检样品0.1ml于上述已包被之反应孔中,置37℃孵育1小时。然后洗涤。(同时做空白孔,阴性对照孔及阳性对照孔)。
3. 加酶标抗体:于各反应孔中,加入新鲜稀释的酶标抗体(经滴定后的稀释度)0.1ml,37℃孵育0.5~1小时,洗涤。
4. 加底物液显色:于各反应孔中加入临时配制的TMB底物溶液0.1ml,37℃10~30分钟。
5. 终止反应:于各反应孔中加入2M硫酸0.05ml。
6. 结果判定:可于白色背景上,直接用肉眼观察结果:反应孔内颜色越深,阳性程度越强,阴性反应为无色或极浅,依据所呈颜色的深浅,以“+”、“-”号表示。也可测OD值:在ELISA检测仪上,于450nm(若以ABTS显色,则410nm)处,以空白对照孔调零后测各孔OD值,若大于规定的阴性对照OD值的2.1倍,即为阳性。
方法二:用于检测未知抗体的间接法:
用包被缓冲液将已知抗原稀释至1~10μg/ml,每孔加0.1ml,4℃过夜。次日洗涤3次。加一定稀释的待检样品(未知抗体)0.1ml于上述已包被之反应孔中,置37℃孵育1小时,洗涤。(同时做空白、阴性及阳性孔对照)于反应孔中,加入新鲜稀释的酶标第二抗体(抗抗体)0.1ml,37℃孵育30~60分钟,洗涤,最后一遍用DDW洗涤。其余步骤同“双抗体夹心法”的4、5、6。
我现在用商品化的试剂盒进行夹心法ELISA测定某抗体的浓度,盒子给定了4个标准品,标准品抗体浓度为70000~1000单位,测定步骤中要求对样品进行1:1000稀释后测定。
但是有的样本按照1:1000稀释后,最终OD值大于70000浓度的标准品的OD值,然后使用ELISACALC软件进行四参数拟合,超过了标准曲线的范围,就算不出来这个样品的浓度,但是如果在1000-70000之间的OD值就可以算出相应浓度。请教大神们,接下来我是否可以对样品进行1:2000,1:4000浓度稀释,算出结果后再×2、×4,算作此样品的浓度呢?还是直接给一个>70000的定性结果?或者可以有能算出来的其他软件?
Ps试剂盒中提及Dilutionlinearity(稀释线性?)为141%,这个是什么意思呢?
多谢!!
大家好,我在做双抗夹心法的elisa实验,已经花了20来天了,然而标准曲线的线性一直做不好,而且不稳定。下面我把具体的实验过程说下,希望大家能给我提个建议,谢谢!
首先我从以下数据中确定了标准品的起始稀释浓度:4.64,46.4,464,4640IU/ml对应的OD为0.55,1.178,1.086,1.168.因此我取46.4IU/ml(300ng/ml)作为标品的起始稀释浓度。然后作了关于单抗,多抗,二抗的合适稀释度的实验,我的数据如下:
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空白0.2290.1920.2120.1540.1390.1730.2360.1770.257
46.4iu/ml1.2421.5271.2691.9641.6281.9592.0051.8802.163
9.28iu/ml0.9680.9501.0131.1360.8731.2461.6971.5861.769
阴性0.1920.1760.2500.1310.1610.1540.1990.2190.244
选取了7号组合单抗,多抗,二抗分别为1ug/ml,1ug/ml,0.5ug/ml。
标品按照以下梯度稀释:46.423.211.65.82.91.450.725iu/ml.以下是我近来做的三组数据,都是相对空白的相对值:
123
46.4iu/ml0.6130.5860.851
23.20.5240.6490.765
11.60.3820.510.663
5.80.3160.3850.567
2.90.2880.3190.37
1.450.2890.1340.303
0.7250.1210.1460.113
空白0.0820.0870.101
阴性0.0820.1050.101
似乎梯度都不明显。我现在疑惑的是我的标品浓度的设置有问题还是之前抗体的浓度选择不合适,请大家多指点,万分感谢!!!