小鼠CaATP酶(CaATPase)ELISA试剂盒说明书

小鼠Ca-ATP酶（Ca-ATPase）酶联免疫吸附测定试剂盒 利用说明书产品编号：E-EL-M0209（本试剂盒仅供体外研究利用、不用于临床诊断！）声明：尊重的客户，感激您选用本公司的产品。本产品选用世界闻名生产厂家的原料，采纳 专业ELISA kit生产技术制造。适用于体外定量检测 /、鼠血清、血浆、组织匀浆或细胞培育 上清液中天然和重组 Ca-ATPas靡度。利用前请认真阅读说明书并检查试剂组分！如有疑问,请及时联系伊莱瑞特生物科技。试剂盒组成：名称-中文名称-英文规格保存条件ELISA酶标板Micro ELISA Plate8X 12 / 8 X6*4 c冻干标准品Reference Standard2/ 1 支*4 c标准品&羊品稀释液Reference Standard & Sample Diluent1 瓶 20mL/12mL*4 c浓缩生物素化抗体Concentrated Biotinylated Detection Ab1 支 120V L/70 g L*4 c生物素化抗体稀释液Diluent for Biotinylated Detection Ab1 瓶 10mL/6mL*4 c浓缩HRPB结合物Concentrated HRP Conjugate1 支 120V L/70 g L*44（避光）酶结合物稀释液Diluent for HRP Conjugate1 瓶 10mL/6mL*4 c浓缩洗涤液（25X ）Concentrated Wash Buffer(25x)1 瓶 30mL/16mL*4 c底物溶液（TMBSubstrate Reagent1 瓶 10mL/6mL*4c (避光)反应终止液Stop Solution1 瓶 10mL/6mL*4 c封板覆膜Plate Sealer5/3 张*产品说明书Product Description1份*: 96T/48T （打开包装后请及时检查所有物品是不是齐全完整）检测原理 :本试剂盒采纳双抗体夹心 ELISA法。用抗小鼠Ca-ATPas啦体包被于酶标板上，实验时标本或标准 品中的Ca-ATPase会与包被抗体结合，游离的成份被洗去。依次加入生物素化的抗小鼠 Ca-ATPase 抗体和辣根过氧化物酶标记白亲和素。抗小鼠Ca-ATPase抗体与结合在包被抗体上的小鼠Ca-ATPase吉合、生物素与亲和素特异性结合而形成免疫复合物，游离的成份被洗去。加入显色底物（TMB）, TMBB辣根过氧化物酶的催化下现蓝色，加终止液后变黄。用酶标仪在450nmM长处测OD直，Ca-ATPase浓度与0岛0值之间呈正比，通过绘制标准曲线求出标本中Ca-ATPase的浓度。标本搜集 :1 .血清：全血标本于室温放置 2小时或4c留宿后于1000X g离心20分钟，取上清即可检测，搜集血液的试管应为一次性的无热原，无内毒素试管。2 .血浆：抗凝剂推荐利用 EDTA.Na2标本搜集后30分钟内于1000X g离心15分钟，取上清即可检测。幸免利用溶血，高血脂标本。3 . 组织匀浆：用预冷的PBS1M, pH=7.4） 冲洗组织，以去除残留血液（匀浆中裂解的红细胞会阻碍测量结果），称重后将组织剪碎。将剪碎的组织与对应体积的PBS （一本按1:9的重量体积比，比如1g的组织样本对应9mLJPBS具体体积可依如实验需要适当调整，并做好记录。推荐在PB部加入蛋白酶抑制剂）加入玻璃匀浆器中，于冰上充分研磨。为了进一步裂解组织细胞，能够对匀浆液进行超声破碎，或反复冻融。最后将匀浆液于5000 Xg离心510分钟，取上清检测。4 .细胞培育上清：取细胞培育上清于1000X g离心20分钟，除去杂质及细胞碎片。 取上清检测。5 .其它生物标本：1000Xg离心20分钟，取上清即可检测（具体处置方式可参考：）6 .标本应清澈透明，悬浮物应离心去除。7 .标本搜集后假设不及时检测，请按一次利用量分装，冻存于 -20C/-8 0c冰箱内，幸免反复 冻融，1-6月内检测，4C保留的应在1周内进行检测。8 .若是您的样本中检测物浓度高于标准品最高值，请依如实际情形，做适当倍数稀释（建议先做预实验，以确信稀释倍数 ）。实验所需自备物品：1 .酶标仪（450nm波长滤光片）2 .高精度移液器，EP1及一次性吸头：l L, 2-20科L, 20-200科L, 200-1000科L3 . 37 c恒温箱，双蒸水或去离子水4 .吸水纸检测前预备工作：1 .请提早20分钟从冰箱中掏出试剂盒，平稳至室温。2 .将浓缩洗涤液用双蒸水稀释 （1:25）。未用完的放回4C。从冰箱中掏出的浓缩洗涤液可能有 结晶，属于正常现象，可用40c水浴微加热使结晶完全溶解后再配制洗涤液（加热温度不要超过50C,利历时洗涤液应为室温）。3 .标准品：加入标准品&样品稀释液1.0mL至冻干标准品中,静置10分钟，待其充分溶解后，轻 轻混匀（浓度为2000pg/mL）。然后依照需要进行倍比稀释（注：不要直接在反映孔中进行倍比稀释）。建议配制成以下浓度：2000、1000、500、250、125、0 pg/mL ,样品稀释液直接作为空白孔 0pg/mL。如配制1000pg/mL标准品：取L 2000pg/mL的上述标准品加入含有L样品稀释液的EPf中，混匀即可，其余浓度依此类推。100 L/孔计），实际配制时应多4 .生物素化抗体工作液：实验前计算当次实验所需用量（以配制100-200wL。利用前15分钟，以生物素化抗体稀释液稀释浓缩生物素化抗体（1:100）成工作浓度。当日利用。5 .酶结合物工作液：实验前计算当次实验所需用量（以 100 L/孔计），实际配制时应多配制100- 200科L。利用前15分钟，以酶结合物稀释液稀释浓缩HRP1结合物（1:100 ）成工作浓度。当日利用。标准品稀释方式图例：（以500pL/管为例，也可依如实际用量来稀释，如 200pL/管）洗涤方式：1 .自动洗板机：每孔加入洗涤液 350 dL，注入与吸出距离60秒。2 .手工洗板：甩尽孔内液体，在干净的吸水纸上拍干，每孔加洗涤液 350 L,浸泡1-2分钟, 吸去（不可触及板壁）或甩掉酶标板内的液体，在厚的吸水纸上拍干。操作步骤：实验开始前，各试剂均应平稳至室温；试剂或样品配制时，均需充分混匀，并尽可能幸免起泡。1 .加样：别离设空白孔、标准孔、待测样品孔。空白孔加样品稀释液100 pL,余孔别离加标准品或待测样品 100 L,注意不要有气泡，加样时将样品加于酶标板底部，尽可能不触及孔壁，轻轻晃动混匀。给酶标板覆膜，37孵育90 分钟。 为保证明验结果有效性，每次实验请利用新的标准品溶液。2 .弃去液体，甩干，不用洗涤。每一个孔中加入生物素化抗体工作液100 dL （在利用前15分钟内配制），酶标板加上覆膜，37温育1 小时。3 .弃去孔内液体，甩干，洗板 3次，每次浸泡1-2分钟，大约350d L/每孔，甩干并在吸水纸上轻拍将孔内液体拍干。4 .每孔加酶结合物工作液 （临用前15分钟内配制）100 dL，加上覆膜，37 c温育30分钟。5 . 弃去孔内液体，甩干，洗板5 次，方式同步骤3。6 .每孔加底物溶液（TMB）100d L,酶标板加上覆膜 37c避光孵育15分钟左右（依如实际显色情形酌情缩短或延长，但不可超过30 分钟。当标准孔显现明显梯度时，即可终止）。7 .每孔加终止液50科L,终止反映，现在蓝色立转黄色。终止液的加入顺序应尽可能与底物溶液的加入顺序相同。8 .当即用酶标仪在 450nm波长测量各孔的光密度（OD值）。应提早打开酶标仪电源，预热仪器，设置好检测程序。9 . 实验完毕后将未用完的试剂按规定的保留温度放回冰箱保留。注意事项 ：1. 保留： 试剂盒中各试剂请按说明书提示合理寄存。在贮存及温育进程中幸免将试剂暴露在强光中。所有试剂瓶盖须旋紧以避免蒸发和微生物的污染，不然可能会显现错误的结果。2. 酶标板：刚开启的酶标板孔中可能会有少量水样物质，此为正常现象，可不能对实验结果造成任何阻碍。3. 加样： 加样或加试剂时，第一个孔与最后一个孔的加样时刻距离若是太大，将会致使不同的“预温育”时刻，从而明显地阻碍到测量值的准确性及重复性。每次的加样时刻最好操纵在10分钟内。推荐设置复孔。4. 温育： 为避免样品蒸发，实验时必需给酶标板覆膜；洗板后应尽快进行下步操作，幸免酶标板处于干燥状态；严格遵守给定的温育时刻和温度。5. 洗涤 ：洗涤进程中反映孔中残留的洗涤液应在吸水纸上拍干，勿将滤纸直接放入反映孔中吸 水。在读数前要注意清除底部残留的液体和手指印，以避免阻碍酶标仪读数。6. 试剂配制：Concentrated Biotinylated Detection Ab 及 Concentrated HRP Conjugate 体积较小，运输进程会使液体沾到管壁或瓶盖，因此利用前1000转/分离心1min,以使附着管壁或瓶盖的液体沉积到管底。取用前，请用移液器警惕吹打4-5 次使溶液混匀。标准品、生物素化抗体工作液、 酶结合物工作液请依照所需用量配制，并利用相应的稀释液配制，不能混淆。 请精准配制标准品及工作液，尽可能不要微量配制（如吸取Concentrated BiotinylatedDetection Ab时，一次不要小于10L）,以幸免由于不准确稀释而造成浓度误差；请勿重复 利用已稀释过的标准品、生物素化抗体工作液、 酶结合物工作液。假设需要分次利用标准品应依照每一次用量分装，将其放在-20-8 0C贮存。幸免反复冻融。7. 显色时刻的操纵：加入底物后请按时观看反映孔的颜色转变（比如每隔5分钟） ，如梯度已很明显，请提早加入终止液终止反映，幸免颜色过深阻碍酶标仪读数。8. 底物： 底物请避光保留，在贮存和温育时幸免强光直接照射。9. 混匀： 充分轻微混匀对反映结果尤其重要，最好利用微量振荡器（ 利用最低频率） ，如无微量振荡器，可在反映前手工轻轻敲击酶标板框混匀。10. 平安： 实验中请穿着实验服并带乳胶手套做好防护工作。专门是检测血液或其他体液标本时，请按国家生物实验室平安防护条例执行。11. 不同批号的试剂盒组份不能混用（洗涤液和反映终止液除外）12. 实验中所用的EPf和吸头均为一次性利用，严禁混用，不然将阻碍实验结果！结果判定 :1 .每一个标准品的OD直减去空白孔的OD直后作图，如设置复孔，那么应取其平均值计算。以标准品的浓度为横坐标，。明为纵坐标，绘出标准曲线。亦能够OD直为横坐标，标准品的浓度为纵坐标，绘出标准曲线。2 .推荐利用专业的曲线制作软件，如 curve expert 1.3,在软件界面既可依照样品0明，由标准曲线查出相应的浓度，乘以稀释倍数；亦可将样品的OD直代入标准曲线的拟合方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。3 .假设标本0明高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数。灵敏度、检测范围、特异性和重复性： 灵敏度：最小可测 pg/mL。 检测范围：-2000pg/mL。 特异性：可检测重组或天然的小鼠Ca-ATPase,且与其它相关蛋白无交叉反映。 重复性：板内，板间变异系数均10%问题分析问题描述可能原因相应对策标准曲线梯度差吸液或加液不准检查移液器及吸头标准品稀释不正确溶解标准品时稍微旋转瓶身，轻轻混匀使粉末完全溶解洗涤不完全保证洗涤时间和洗涤次数及每孔的加液量显色很弱或无色孵育时间太短保证充足的孵育时间实验温度不正确使用推荐的实验温度试剂体积不够或漏加检查吸液及加液过程，保证所有试剂按顺序足量添加稀释不正确读数数值低酶标仪设置不正确在酶标仪上检查波长及滤光片设置提前打开酶标仪预热曲线偏差大加液不正确检查加液情况背景值高检测抗体的工作浓度过高使用推荐的稀释倍数酶标板洗涤不完全重新阅读操作手册，保证清洗完全；如果用自动洗板机，请检查所有的出口是否有堵塞洗液有污染配制新鲜的洗液灵敏度低ELISA试剂盒保存不当按说明书要求保存相关试剂读数前未终止OD读数前应在每孔中加入终止液说明1 .限于现有条件及科学技术水平，尚不能对所有原料进行全面的鉴定分析，本产品可能存在 必然的质量技术风险。2 .最终的实验结果与试剂的有效性、实验者的相关操作和那时的实验环境紧密相关，请务必预备充沛的待测样品。3 .只有全数利用 日abTM试剂才能保证检测成效，不能混用其他制造商的产品。只有严格遵守 Elab TM试剂的实验说明才会取得最正确的检测结果。4 .有效期：6个月。5 .本操作说明一样适用于 48T试剂盒。Mouse Ca-ATPase (Calcium ATPase) ELISA KitProduct DescriptionCatalog No: E-EL-M0209(FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGONOSIS !)Dear customer, Thank you for choosing our products. This product is produced using rawmaterials from world-renowned manufacturer, and professional manufacturing technology of ELISA kits. Please read the instructions carefully before use and check all the reagent compositions! If in doubt, please contact Elabscience Biotechnology Co., Ltd.Intended useThis immunoassay kit allows for the in vitro quantitative determination of Mouse Ca-ATPase concentrations in serum, plasma and other biological fluids.Kit Components :ItemSpecificationsStorageMicro ELISA Plate8X12 or 8 X6 *4 CReference Standard2/ 1vial *4 CReference Standard & Sample Diluent1vial 20mL/12mL *4。CConcentrated Biotinylated Detection Ab1via l120 dL/70 w L *4 CDiluent for Biotinylated Detection Ab1vial 10mL/6mL*4 CConcentrated HRP Conjugate1vial 120 w L/70 L *4 C (shading light)Diluent for HRP Conjugate1vial 10mL/6mL *4 CConcentrated Wash Buffer(25X)1vial 30mL/16mL *4 CSubstrate Reagent1vial 10mL/6mL *4 C (shading light)Stop Solution1vial 10mL/6mL *4 CPlate Sealer5/3pieces *Product Description1 copy*: 96T/48TTest principleThis ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Ca-ATPase Standards or samples are then added to the appropriate micro ELISA plate wells and combined to the specific antibody. Then a biotinylated detection antibody specific for Ca-ATPase and Avidin-Horseradish Peroxidase (HRP) conjugate is added to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Ca-ATPase, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turn yellow. The optical density (OD) is measured spectrophotometri cally at a wavelength of 450 nm 2 nm. The OD value is proportional to the concentration of Ca-ATPase. You can calculate the concentration of Ca-ATPase in the samples by comparing the O.D. of the samples to the standard curve.Sample collection and storageSerum - Allow samples to clot for 2 hours at room temperature or overnight at 4Cbeforecentrifugation for 20 minutes at approximately 1000x g. Collect the supernatant andcarry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.Plasma - Collect plasma using EDTA.Na2or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 Xg at 2- 8 C within 30 minutes of collection. Collect thesupernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.Tissue homogenates: For general information, hemolysis blood may affect the result, so1M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then minced to small pieces which will be homogenized in PBS(the volume depends on the weight of the tissue. 9mL PBS would be appropriate to 1 gram tissue pieces.Some proteaseinhibitor is recommended to add into the PBS.) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter orsubject it to freeze-thaw cycles. The homogenates are then centrifugated for 5minutes at 5000 x g to get the supernate.Cell culture supernate- Centrifuge supernate for 20 minutes to remove insolubleimpurity and cell debrisat 1000 x g at 2 - 8 C. Collect the clear supernate and carryout the assay immediately.Other biological fluids- Centrifuge samples for 20 minutes at 1000 Xg at 2- 8 C.Collect the supernatant and carry out the assay immediately. (You can refer to our website for detailed processing method: )Sample preparation - Samples should be clear and transparent and be centrifuged to remove suspended solids.Note: Serum and plasma to be used within 7 days when stored at 2-8 C , otherwise samples must be divided and stored at -20 C ( 1 month) or -80 C ( 6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. If the sample concentration is higher than the maximum standard value, please dilute it with appropriate factor according to the actual situation. (A pre-test is recommended to determine the dilute factor)Other supplies requiredMicroplate reader with 450nm wavelength filterHigh-precision transferpettor, EP tubes and disposable pipette tips 37 C Incubator, Deionized or distilled water.Absorbent paperReagent preparationBring all reagents to room temperature before use.Wash Buffer - Dilute 30mL of Concentrated Wash Buffer into 750 mL of Wash Buffer withdeionized or distilled water. Put unused solution back at 4 C. If crystals have formed in the concentrate, you can warm it with 40 C water bath (Heating temperature shouldnot exceed 50 C) and mix it gently until the crystals have completely dissolved. The solution should be cooled to room temperature before use.Standard - Reconstitute the Standard with 1.0mL of Sample Diluent , let it stand for10minutes until it dissolved fully. This reconstitution produces a stock solution of2000pg/mL. Then make serial dilutions as needed (Making serial dilution in the wellsdirectly is not permitted). The recommended concentrations are as follows: 2000、1000、 500、250、125、0pg/mL . As if you want to make standard solution at the concentration of 1000pg/mL, you can take L the standard at 2000pg/mL, add it to an L sample dilution, and mix it. The procedures of making the remaining concentrations are all the same. The undiluted standard serves as the highest standard (2000pg/mL). The Sample Diluent serves as the zero (0pg/mL).(500L/tube , for example. Can also be diluted according to the actual amount, such as 200 P L/tube)200010005002501250 pg/mLBiotinylated Detection Ab Calculate the required amount before experiment (100科 L/well). In actual preparation you should prepare10020O(i L more. Dilute theconcentrated Biotinylated Detection Ab to the working concentration using Diluent for Biotinylated Detection Ab (1:100).Concentrated HRP Conjugate Calculate the required amount before experiment (100L/well). In actual preparation you should prepare 100200科 L more. Dilute theConcentrated HRPConjugate to the working concentration using Diluent for Concentrated HRP Conjugate (1:100).Washing Procedure:1. Automated washer: add 350 科 L wash buffer into each well, the interval between injection and suction should be set about 60s.2. Manual wash: add 350 科 L wash buffer into each well, soak it for 12minutes, suck(no inside wall touching) or get rid of liquid within the micro ELISA plate and pat itdry on thick clean absorbent paper.Assay procedureAllow all reagents to reach room temperature All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming.1. Add Sample: Add 100 科 Lof Standard, Blank, or Sample per well. The blank well isadded with sample diluent. Solutions are added to the bottom of micro ELISA platewell, avoid inside wall touching and foaming to the best of your ability. Mix itgently. Cover the plate with sealer we provided. Incubate for 90 minutes at 37C .2. Biotinylated Detection Ab: Removethe liquid of each well, don t wash. Immediately add 100L of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37 C .3. Wash: Aspirate each well and wash, repeating the process three times. Washby filling each well with Wash Buffer (approximately 350 科 L) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.4. HRPConjugate: Add 100L of HRPConjugate working solution to each well. Cover with the Plate sealer. Incubate for 30minutes at 37 C.5. Wash: Repeat the wash process for five times as conducted in step 3.6. Substrate: Add 100 科 L of Substrate Solution to each well. Cover with a new Platesealer. Incubate for about 15 minutes at 37 C. Protect the plate from light. Thereaction time can be shortened or extended according to the actual color change, but not more than 30minutes. Whenapparent gradient appeared in standard wells, you can terminate the reaction.7. Stop : Add 50 科 Lof Stop Solution to each well. Colorturn to yellow immediately.The adding order of stop solution should be as the same as the substrate solution.8. ODMeasurement: Determine the optical density (OD value) of each well at once, using a microplate reader set to 450nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.9. After experiment, put all the unused reagents back into the refrigerator according to the specified storage temperature respectively until their expiry.Important Note:1. Storage : All the reagents in the kit should be stored following the instructions.Exposure of reagents to strong light should be avoided in the process of incubation and storage. All the taps of reagents should be tightened to prevent evaporation and microbial contamination, or erroneous results may occur.2. ELISA Plate : Little water-like substance may appear in the ELISA Plate just opened, this is normal and will not have any impact on the experiment results.3. Add Sample: The interval of sample adding between the first well and the last wellshould not be too long, otherwise will cause different pre-incubation time, whichwill significantly affect the experiment s accuracy and repeatability. The interval controlled within 10minutes is good. Parallel measurement is recommended.4. Incubation : To prevent evaporation, proper adhesion of platesealers duringincubation steps is necessary. Do not allow wells to sit uncovered for extendedperiods between incubation steps. Do not let the strips dry at any time during theassay. Strict compliance with the given incubation time and temperature.5. Washing: The wash procedure is critical. Insufficient washing will result in poorprecision and falsely elevated absorbance readings Residual liquid in the reaction wells should be pat dry against absorbent paper in the washing process. But don tput absorbent paper into reaction wells directly. Note that clear the residual liquid and fingerprint in the bottom before measurement, so as not to affect the microtiter plate reader.6. Reagent Preparation : As the volume of Concentrated Biotinylated Detection Ab and Concentrated HRPC onjugate is very small, liquid may adhere to the tube wall or tube cap when being transported. You better hand-throw it or centrifugal it for 1 minute at 1000rpm. Please pipette the solution for 4-5 times before pippeting. Please carefully reconstitute Standards, working solutions of Biotinylated Detection Aband HRP Conjugate according to the instructions. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than10L for once pipetting. Do not reuse standard solution, working solution ofBiotinylated Detection Ab and HRP Conjugate, which have been diluted. If you needto use standard repeatedly, you can divide the standard into small pack accordingto the amount of each assay, keep them at -20-80 C and avoid repeated freezingand thawing.7. Reaction Time Control: Please control reaction time strictlyfollowing this productdescription!8. Substrate: Substrate Solution is easily contaminated. Please protect it from light.9. Mixing:You d better use microoscillator at the lowest frequency, as sufficientand gentle mixing is particularly important to reaction result. If there is no microsocillator available, you can knock the ELISA plate frame gently with your finger before reaction.10. Security: Please wear lab coats and latex gloves for protection. Especially detectingsamples of blood or other body fluid,please perform following the nationalsecurity columns of biological laboratories.11. Do not use component from different batches of kit(washing buffer and stop solutioncan be an exception)12. To avoid cross-contamination, change pipette tips between adding of each standardlevel, between sample adding, and between reagent adding. Also, use separate reservoirs for each reagent. Otherwise, the results will be inaccurate!Calculation of resultsAverage the duplicate readings for each standard and samples and subtract the average zero standard optical density. Create a standard curve by plotting the mean OD value for each standard on the y-axis or x-axis against the concentration on the x-axis or y-axis and draw a best fit curve through the points on the graph. It is recommended to use some professional software to do this calculation, such as curve expert 1.3. In the software interface, a best fitting equation of standard curve will be calculated using OD values and concentrations of standard sample. The software will calculate the concentration of samples after entering the OD value of samples. Also, you can enter the corresponding fitting equation and OD value of samples into Excel to get the concentration of samples. If samples have been diluted, the concentration calculatedfrom the standard curve must be multiplied by the dilution factor. If the ODof the sample surpasses the upper limit of the standard curve, you should re-testit after appropriatedilution.The actual concentration is the calculated concentrationmultiplied dilutionfactor.SensitivityThe minimum detectable dose of Mouse Ca-ATPase is pg/mL (The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero).Detection Ran