大鼠(Rat)血管內(nèi)皮鈣粘蛋白(VE-cadherin)-NEWA

1、精品文檔本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷地分離。大鼠（ Rat ）血管內(nèi)皮鈣粘蛋白（ VE-cadherin ）2.血漿： EDTA、檸檬酸鹽或肝素抗凝。 3000轉(zhuǎn)離心 30 分鐘取上ELISA 檢測試劑盒清。使用說明書3.細(xì)胞上清液： 3000 轉(zhuǎn)離心 10 分鐘去除顆粒和聚合物。檢測原理4.組織勻漿：將組織加入適量生理鹽水搗碎。3000 轉(zhuǎn)離心 10 分試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往鐘取上清。預(yù)先包被血管內(nèi)皮鈣粘蛋白（ VE-cadherin ）抗體的包被微孔中，依5.保存：如果樣本收集后不及時(shí)檢測， 請按一次用量分裝， 凍存于次加入標(biāo)本、標(biāo)準(zhǔn)品、

2、 HRP標(biāo)記的檢測抗體，經(jīng)過溫育并徹底洗滌。-20，避免反復(fù)凍融，在室溫下解凍并確保樣品均勻地充分解凍。用底物 TMB 顯色， TMB 在過氧化物酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸自備物品的作用下轉(zhuǎn)化成最終的黃色。顏色的深淺和樣品中的血管內(nèi)皮鈣粘1.酶標(biāo)儀（ 450nm ）蛋白（ VE-cadherin ）呈正相關(guān)。用酶標(biāo)儀在 450nm波長下測定吸2.高 精 度 加 樣 器及 槍 頭 ： 0.5-10uL 、 2-20uL 、 20-200uL 、光度（ OD 值），計(jì)算樣品濃度。200-1000uL樣品收集、處理及保存方法3.37 恒溫箱1. 血清：使用不含熱原和內(nèi)毒素的試管， 操作過程中避免任

3、何細(xì)胞操作注意事項(xiàng)刺激，收集血液后， 3000 轉(zhuǎn)離心 10 分鐘將血清和紅細(xì)胞迅速小心1.試劑盒保存在 2-8 ，使用前室溫平衡 20分鐘。從冰箱取出的隨意編輯精品文檔濃縮洗滌液會(huì)有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶完全溶解后再使用。2. 實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中， 密封（低溫干燥）保存。3. 濃度為 0 的 S0 號(hào)標(biāo)準(zhǔn)品即可視為陰性對照或者空白；按照說明書操作時(shí)樣本已經(jīng)稀釋 5 倍，最終結(jié)果乘以 5 才是樣本實(shí)際濃度。4. 嚴(yán)格按照說明書中標(biāo)明的時(shí)間、加液量及順序進(jìn)行溫育操作。5. 所有液體組分使用前充分搖勻。試劑盒組成名稱96 孔配置48 孔配置備注微孔酶標(biāo)板12 孔

4、15;8條12 孔×4條無標(biāo)準(zhǔn)品0.3mL*6管0.3mL*6管無樣本稀釋液6mL3mL無檢測抗體 -HRP10mL5mL無20 ×洗滌緩沖液25mL15mL按說明書進(jìn)行稀釋底物 A6mL3mL無底物 B6mL3mL無終止液6mL3mL無封板膜2 張2 張無說明書1 份1 份無自封袋1 個(gè)1 個(gè)無注：標(biāo)準(zhǔn)品（S0-S5 ）濃度依次為：0 、0.5 、 1 、 2 、 4 、 8g/mL試劑的準(zhǔn)備20 ×洗滌緩沖液的稀釋： 蒸餾水按 1 ：20 稀釋，即 1 份的 20 ×洗滌緩沖液加 19 份的蒸餾水。洗板方法1. 手工洗板：甩盡孔內(nèi)液體，每孔加滿洗滌液

5、，靜置 1min 后甩盡孔內(nèi)液體，在吸水紙上拍干，如此洗板 5 次。2. 自動(dòng)洗板機(jī)：每孔注入洗液 350 L，浸泡 1min ，洗板 5 次。操作步驟1.從室溫平衡 20min后的鋁箔袋中取出所需板條，剩余板條用自隨意編輯精品文檔封袋密封放回 4 。2. 設(shè)置標(biāo)準(zhǔn)品孔和樣本孔， 標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品 50 L；3. 樣本孔先加待測樣本 10 L，再加樣本稀釋液 40 L；空白孔不加。4. 除空白孔外，標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶（HRP ）標(biāo)記的檢測抗體100 L，用封板膜封住反應(yīng)孔，37 水浴鍋或恒溫箱溫育60min 。5.棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置1

6、min ，甩去洗滌液，吸水紙上拍干，如此重復(fù)洗板5 次（也可用洗板機(jī)洗板） 。試劑盒性能6.每孔加入底物 A 、B 各 50 L，37 避光孵育 15min 。1. 準(zhǔn)確性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R 值，大于等于7.每孔加入終止液50 L，15min內(nèi)，在 450nm波長處測定各孔0.9900 。的OD 值。2.靈敏度：最低檢測濃度小于0.1 g/mL 。結(jié)果判斷3.特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)。繪制標(biāo)準(zhǔn)曲線：在Excel 工作表中，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對應(yīng)4.重復(fù)性：板內(nèi)、板間變異系數(shù)均小于15% 。OD 值作縱坐標(biāo)， 繪制出標(biāo)準(zhǔn)品線性回歸曲線， 按曲線方程計(jì)算各樣5

7、.貯藏： 2-8 ，避光防潮保存。本濃度值。6.有效期： 6 個(gè)月隨意編輯免責(zé)聲明1. 試劑盒僅供研究使用，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所產(chǎn)生的一切后果，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。2. 嚴(yán)格按照說明書操作，實(shí)驗(yàn)者違反說明書操作，后果由實(shí)驗(yàn)者承擔(dān)。精品文檔color is measured at 450 nm using a spectrophotometer. In order tomeasuretheconcentrationofVE-cadherininthesample,thisVE-cadherinELISAKitincludesa setofcalibrationstanda

8、rds.Thecalibration standards are assayed at the same time as the samples andallowtheoperatortoproducea standardcurveofOpticalDensityversus VE-cadherin concentration. The concentration of VE-cadherin inthe samples is then determined by comparing the O.D. of the samplesFOR RESEARCH USE ONLY.to the sta

9、ndard curve.NOT FOR USE IN DIAGNOSTIC PROCEDURES.Sample collection and storagesSerum - Use a serum separator tube and allow samples to clot for 30Rat VE-cadherin ELISA Kit instructionminutes before centrifugation for 10 minutes at approximately 3000 × g.Intended useRemove serum and assay immedi

10、ately or aliquot and store samples atThis VE-cadherin ELISA kit is intended Laboratory for Research use only- 20 or - 80 .Avoid repeated freeze-thaw cyclesand is not for use in diagnostic or therapeutic procedures.TheStopPlasma- Collect plasma using EDTA or heparin as an anticoagulant.Solution chang

11、es the color from blue to yellow and the intensity of theCentrifugesamples for 30 minutesat 3000 × g at 2 - 8 within 30隨意編輯精品文檔minutes of collection. Store samples at -20 or - 80 . Avoid repeatedfreeze-thaw cycles.Cell culture supernates and other biological fluids- Removeparticulatesby centrif

12、ugationand assay immediatelyor aliquot andstore samples at - 20 or - 80 . Avoid repeated freeze-thaw cycles.Note:The samples shoule be centrifugated dequately and nohemolysis or granule was allowed.Materials required but not supplied1. Standard microplate reader(450nm)2. Precision pipettes and Dispo

13、sable pipette tips.3. 37 incubatorPrecautions1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Useonly the reagents supplied by manufacturer.2. Do not remove microplate from the storage bag until needed. Unused strips shou

14、ld be stored at 2- 8 °Cin their pouch with the desiccant provided.3. Mix all reagents before using.Removeallkitreagentsfromrefrigeratorandallowthemtoreachroom temperature ( 20-25°C)Materials supplied9648NamedeterminationsdeterminationsMicroelisa stripplate12*8strips12*4stripsStandard0.3ml*

15、6tubes0.3ml*6tubesSample Diluent6.0ml3.0mlHRP-Conjugate10.0ml5.0mlreagent20X Wash solution25ml15ml隨意編輯精品文檔Chromogen Solution6.0ml3.0mlAChromogen Solution6.0ml3.0mlBStop Solution6.0ml3.0mlClosure plate22membraneUser manual11Sealed bags11recommendedthatallStandardsand Samples be added in duplicateto t

16、he Microelisa Stripplate.2. Add standard: Set Standard wells, testing sample wells. Addstandard50 l to standard well .3.AddSample: Add testing sample 1 0 lthen addSample Diluent40 l to testing sample well ; Blank well doesn t add anyting.4.Add10 0 lof HRP-conjugate reagent to each well, cover with a

17、nadhesive stripand incubate for 60 minutes at 37°C.5. Aspirate each well and wash, repeating the process four times for aNote: Standard (S0 S5) concentration was followed by:0,0.5,1,2,4,8total offivewashes. WashbyfillingeachwellwithWashSolutiong/ml(400 l)usingasquirtbottle,manifolddispenserorau

18、towasher.Reagent preparationCompleteremovalofliquidateach step isessentialtogood20 ×wash solution:Dilute with Distilled or deionized water 1:20.performance. After the last wash, remove any remaining Wash SolutionAssay procedureby aspiratingor decanting.Inverttheplateandblotitagainstclean1. Prep

19、are all r ea g e n t s before starting assay procedure.It ispaper towels.隨意編輯精品文檔6.Add chromogen solution A 50 l and chromogen solution B 50eachwell.Gentlymixandincubatefor15 minutesat37 °C.Protectfrom light.7. Add 50 Stopl Solution to each well. The color in the wells shouldchangefrombluetoyel

20、low.If thecolorin thewellsis greenor thecolor change does notappear uniform, gently tap the plate to ensure thorough mixing.8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.Calculation of results1.Thisstandardcurveisusedtodeterminetheamountinanunknown sa

21、mple. The standard curve is generated by plotting theaverageO.D.(450nm)obtainedforeachofthesixstandard2. First, lcalculateto the mean O.D. value for each standard and sample.AllO.D. values, are subtractedbythemean valueofthezerostandard before result interpretation. Construct the standard curveusing graph paper or statistical software.3.Todeterminetheamountineach