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View additional product information for Cell Lysis Buffer II - FAQs (FNN0021)
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以下是此问题的可能原因和解决方案：
o 检查协议设置（确保选择正确的 DD 设置）。
o 检查鞘液液位并清空废液。
o 在获取板之前，在 Luminex 仪器上运行校准和验证珠。
以下是可能的原因和问题的解决方案：
•这通常表明微珠已经光漂白。这个问题也可能由于将微珠暴露于有机溶剂中引起。不幸的是，必须重复该检测，因为微珠不能恢复。必须保护珠子免受光和有机溶剂的影响。
•或者，仪器可能在其测量中关闭，或者可能存在校准问题。致电制造商进行服务预约。
这表示最后一步使用了不正确的缓冲液。必须使用试剂盒中提供的洗涤液洗涤微珠并在微珠上样至Luminex仪器之前将其重悬。溶液的渗透压将影响微珠的尺寸，而微珠尺寸的任何变化都将改变仪器的检测。
以下是一些建议：
o 在获取板之前，在 Luminex 仪器上运行校准和验证珠。
o 查看仪器设置并确保它们适合正在运行的检测（调整针高，确保选择正确的珠门和正确的 DD 设置）。
o 在仪器上采集前摇动板以重新悬浮珠子。
o 将珠子涡旋 30 秒，然后再将它们添加到板中。
o 清洗：在清空板之前，不要忘记将板放在手持式磁性洗板机上约 2 分钟。
这种模式表明样本基质效应。以下是一些建议：
o 确认样品已澄清且不含碎片和脂质（建议离心 5-10 分钟）。
o 确认血清、血浆样品的样品与测定稀释剂的比例至少为 1:1。对于细胞裂解物或组织匀浆，确认样品已在测定缓冲液中适当稀释，以将裂解缓冲液中的去污剂浓度降低至≤0.01%。对于其他样品类型，可能需要进一步的样品优化。
以下是一些建议：
o 您感兴趣的蛋白质水平可能低于检测的检测限。高灵敏度多重试剂盒可用于大多数细胞因子。
o 验证您的标准曲线（寻找平台、异常曲线拟合、异常值）。
以下是一些建议：
o 可能需要样品优化：用合适的稀释剂稀释样品并重新运行。
o 验证您的标准曲线（寻找平台、异常曲线拟合、异常值）。
下述方案已经应用于多种人类和小鼠细胞系。您应该为您自己的应用优化细胞提取操作。
细胞裂解操作
非贴壁细胞：
低速离心以沉淀细胞。从沉淀中移去培养基，用冰冷的PBS洗涤两次。去除PBS，将细胞沉淀重悬于细胞裂解缓冲液中（推荐细胞裂解液浓度为2-5 mg/mL）。在冰上孵育15分钟，偶尔涡旋。将裂解物转移至微量离心管中，并在2-8℃下以14000rpm离心10分钟。 将澄清的裂解液等分至干净的微量离心管中，并测定总蛋白浓度。
贴壁细胞：
从细胞中去除培养基，用冰冷的PBS洗涤两次。去除PBS，加入细胞裂解缓冲液（推荐细胞裂解液浓度为2-5μg/mL），并在冰上孵育15分钟。收集细胞裂解液，并将其转移至微量离心管中。在2-8℃下以14000rpm离心10分钟。 将澄清的裂解液等分至干净的微量离心管中，并测定总蛋白浓度。裂解液应在-80°C下冷冻储存，或在收集后立即进行分析。冷冻样品避免多次反复冻融。分析前，将样品完全解冻、充分混合并通过离心（14000rpm，10分钟）来澄清，以防止堵塞过滤板。
推荐的细胞裂解缓冲液：
NP40细胞裂解缓冲液（货号FNN0021）
注意：使用NP40裂解缓冲液制备的裂解液必须在检测之前稀释至少5倍。
或者使用下述配方进行配置：50 mM Tris，pH 7.4，50 mM NaF，260 mM NaCl，1 mM Na3VO4，5 mM EDTA，0.02% NaN3，1% Nonidet P40
NP40细胞裂解缓冲液（不含蛋白酶抑制剂混合物和PMSF）在2-8°C下的稳定期为2-3周，或在-20°C下分装保存6个月。
使用前在NP40细胞裂解缓冲液中新鲜添加：
•1 mM PMSF（DMSO中的0.3 M原液）
•蛋白酶抑制剂混合物（Sigma，货号P-2714）
替代细胞提取缓冲液
细胞提取缓冲液（货号FNN0011）
注意：使用细胞提取缓冲液制备的裂解液必须在检测之前稀释至少10倍。
或者
10 mM Tris，pH 7.4
2 mM Na3VO4
100 mM NaCl
1% Triton X-100
1 mM EDTA
10% 甘油
1 mM EGTA
0.1% SDS
1 mM NaF
0.5% 脱氧胆酸盐
20 mM Na4P2O7
细胞提取缓冲液（不含蛋白酶抑制剂混合物和PMSF）在2-8°C下的稳定期为2-3周，或在-20°C下等分保存6个月。
使用前在细胞提取缓冲液中新鲜添加：
•1 mM PMSF（DMSO中的0.3 M原液）
•蛋白酶抑制剂混合物（Sigma，货号P-2714）
我们没有在内部专门测试唾液样本，因此这些说明只是我们的建议。唾液含有几种蛋白水解酶。将样品离心并确保不要将任何细胞材料或碎片移入测定板是很重要的。我们建议在样品中添加一些抗蛋白酶（例如，trasylol 或 aprotinine，10 至 50 U/mL）以保护蛋白质免受酶降解。然后，您可以在 4 摄氏度以 1000 x g 的速度将样品离心 10 分钟以去除颗粒。立即使用或在 -80 摄氏度下储存等分试样。避免多次冻融循环。
隔离牙齿周围的部位，将一张牙周滤纸插入牙齿周围的牙龈袋中，持续30秒。取出滤纸，在室温下使用50μL PBS提取4次，每次5分钟。每次提取物可以组合和单独进行分析。在应用于检测之前，用检测稀释液稀释2倍。
收集后，应立即将宫颈取样海绵放置于冰上。样品应在-20°C下储存长达一周，然后储存于-80°C直至需要检测准备。解冻后，应将海绵称重并放入Eppendorf管中，每次转移均要使用乙醇处理过的镊子进行。向每个管中加入200μL冰冷的提取缓冲液（配方见下文），并在4℃下孵育过夜。 然后将海绵和提取缓冲液转移到具有0.2μm醋酸纤维素过滤器的微量离心管中，并在4℃下以13000rpm离心10分钟。 然后测试洗脱液中的细胞因子表达。
提取缓冲液
50 mM HEPES，pH 7.5
1 mM Na3VO4
150 mM NaCl
1 mM NaF
1 mM EDTA
0.1% Tween 20
25 mM EGTA
10% 甘油
支气管肺泡灌洗（BAL）液应收集于无菌注射器中，并在分析之前持续保存于冰上。或者，可以将BAL等分至需用样品大小并冷冻（以便只会经历一次冻融）。分析前，所有样品需要通过离心（14000rpm，10分钟）和/或过滤来澄清，以防止堵塞过滤板。在应用于检测板之前，用检测稀释液稀释2倍。
分析前，所有样品需要通过离心（14000rpm，10分钟）和/或过滤来澄清，以防止堵塞过滤板。然而，CSF具有相对较低的粘度，并且除非存在感染状态（WBC的丰度），否则其不需要澄清并可以直接应用于检测。使用神经科学缓冲液试剂盒（货号LNB0001）中提供的检测稀释液稀释2倍。
将关节液收集到非肝素化管中，并在样品采集的30分钟内以1000×g离心10分钟。在进行随后的分析之前，关节液的无细胞部分应储存在-80℃。分析前，所有样品需要通过离心（14000rpm，10分钟）和/或过滤来澄清，以防止堵塞过滤板。在加入检测之前，用检测稀释液以1：1稀释样品。参考文献：Raza K等人（2005） 关节炎研究与治疗7（4）: R784–R795。
我们建议遵循下面提供的的方案，这是基于组织提取试剂I（货号FNN0071）开发并显示了ELISA和Luminex技术之间的良好相关性。该方法已应用于多种组织类型。但是，我们建议您针对所使用的每种组织样品进行优化。可以使用类似的提取试剂/裂解缓冲液。
1.在使用前将蛋白酶抑制剂添加到组织提取试剂中。
2.称量组织样品。
3.每1克组织加入10 mL组织提取试剂。
4.匀化组织。
5.以10000rpm离心样品5分钟以沉淀组织碎屑。
6.收集上清液。按照试剂盒中提供的操作步骤进行适当的稀释；这是为了防止/最小化提取液或裂解缓冲液中存在的去垢剂对抗体 - 抗原结合的潜在抑制。通常，组织匀浆或细胞裂解物（取决于所用的裂解缓冲液）需要稀释5至10倍，以将去垢剂浓度降低至≤0.01％。然而，基于目标细胞因子/蛋白质的浓度，不同的试剂盒/样品可能需要用检测稀释液或标准品稀释液进一步稀释。
注意：如果要储存样品，请将其等分并冷冻于-80°C。避免多次反复冻融。
细胞应处于对数生长期。在适当的细胞培养瓶中根据需要刺激细胞。使用无菌技术，用移液器吸移所需体积的条件细胞培养基，并将培养基转移到干净的聚丙烯微量离心管中。在冷冻微量离心机中，4℃ 14000rpm将培养基离心10分钟，以去除细胞或细胞碎屑。将澄清的培养基等分至干净的聚丙烯微量离心管。这些样品即可用于检测。或者，可将澄清的培养基样品等分并储存在-80℃用于将来分析。避免多次反复冻融。将冷冻样品置于冰上使其解冻，并应在解冻后立即进行测试。分析前，解冻的样品应立即在冷冻微量离心机中通过离心（4℃ 14000rpm离心10分钟）澄清，以防止堵塞Luminex探针和/或过滤板。遵循试剂盒提供的检测程序进行适当的稀释。
在冷冻离心机中以2000×g离心10分钟，将细胞从血浆样品中分离。需要在该力下离心以去除样品中的血小板。用无菌巴斯德移液器将上清液转移到冷却的干净聚丙烯管中。处理时使样品保持在2-8°C。
如果血浆有待日后分析，请将其分装至聚丙烯微量离心管并储存在-80℃。 避免多次反复冻融。分析之前，将样品置于冰上，使其解冻。所有血浆样品应在分析前在冷冻微量离心机中通过离心（4℃下以14000rpm转速离心10分钟）澄清。遵循试剂盒提供的检测程序进行适当的稀释。
应使用无热原/无内毒素的试管采集血清样品。将全血在室温下静置15-30分钟，使其凝固。在4℃冷冻离心机中以1000-2000×g离心10分钟以分离细胞。用无菌巴斯德移液器将上清液转移到冷却的干净聚丙烯管中。处理时使样品保持在2-8°C。
如果血清有待日后分析，请将其以0.5 mL体积分装后储存在-80℃。 避免多次反复冻融。如果可能，避免使用溶血或有脂血症的血清。我们建议，分析前，在解冻后立即通过离心（14000rpm，10分钟）和/或过滤来澄清样品，以防止堵塞过滤板和/或探针。按照试剂盒提供的检测程序进行适当的稀释。
ProcartaPlex 多重检测基于 Luminex xMAP 技术，提供了一个多功能平台，为用户提供了更大的灵活性和更多的分析物检测选项。无论您是测试单一分析物还是多种分析物，ProcartaPlex 多重分析都使用高效、易于遵循的方案提供准确的分析性能。这些检测中的每一个都经历了我们为 ELISA 进行的相同的开发、验证、制造和质量控制标准化。每批 ProcartaPlex 多重检测和 ELISA 检测都经过适当的样本类型（即物种特异性血清、血浆和细胞培养上清液）的完全验证，并且每批都根据以下性能特征进行评估：
o特异性 - 筛选每个分析物以确保在多重测试中与其他分析物没有明显的交叉反应
o灵敏度 - 对每个分析物的功能灵敏度（与背景的区别）和检测下限 (LLOD) 进行评估
o精密/准确度多重检测具有良好的检测内精密度 (<10% CV)、检测间精密度 (<10% CV) 和批次间一致性 (<20% CV)；这些值与大多数 ELISA 测试相当或更好
ProcartaPlex 多重检测定期针对匹配的 ELISA 进行测试。因此，您可以轻松地从 ProcartaPlex 检测切换到 ELISA，反之亦然，结果可靠。我们的大多数 ProcartaPlex 检测使用与我们传统的基于平板的 ELISA 相同的抗体对，从而导致两种检测之间的高度相关性 (R2 > 0.9)。
ELISA是一种可以在复杂样品中特异性定量单个蛋白质的简单而有效的方法。通过使用高质量的单抗体或双抗体夹心技术实现ELISA的选择性，并通过使用经校准的标准品实现精确定量。ELISAs可以检测低丰度的蛋白质，可以在96孔板中进行，并且仅需60分钟的手动操作时间。此外，ELISA获得的结果通常具有高度可重复性。
虽然ELISA已被作为蛋白质分析的标准方法，但是能够在单个样品中同时测量多种分析物的多重检测方法解决了许多缺陷：
•ELISA检测给定样品时，一次仅测量一种分析物，限制了研究人员日益增加的在研究中测量多种靶标的需求。
•所研究的许多样品可用体积较少可能限制可以进行分析的次数。在提供有限样品体积的小动物研究、儿科测试和微孔板分析中尤其如此。能在单个小体积样品中分析多种分析物可以更有效地使用每种样品。
•当对多次ELISA对分析物的检测结果进行比较时数据解读可能会遇到困难。因为每次检测使用的是不同的分装样本，而且每次检测都可能会有系统误差，这些会导致准确性和精度下降。
•许多分析物需要具有较宽动态范围的检测试剂盒，以避免重复测试或出现超出检测范围的情况。多重检测可以设计为具有针对所有分析物的宽动态范围，或根据各种预期分析物浓度专门定制的范围。
Luminex xMAP技术基于聚苯乙烯或顺磁微球或微珠，其内部用不同强度的红色和红外荧光团标记。每个带颜色的微珠都有一个唯一的编号，称为“微珠区域”，以区分不同的微珠。对于Invitrogen多重免疫检测试剂盒，每个微珠组用一种捕获抗体包被以用于一种特定分析物的检测。然后可将多种分析物特异性微珠在96孔检测板的单个孔中混合，使用其中一种Luminex仪器同时进行多种靶标的检测和定量。我们提供使用聚苯乙烯珠或顺磁磁珠进行的多重检测试剂盒。观看视频(https://www.youtube.com/watch?v=kEdLGcGXrs4&feature=youtu.be)，了解如何在Luminex仪器平台上使用Invitrogen多重微珠试剂盒同时检测多种蛋白质。
具有确定光谱特性的微珠与蛋白质特异性捕获抗体偶联，并与样品（包括已知蛋白质浓度的标准品、对照样品和测试样品）一起添加到微孔板的孔中。靶蛋白在2小时的孵育过程中与捕获抗体结合。洗涤微珠后，加入蛋白质特异性生物素化的检测抗体，并与微珠一起孵育1小时。然后，去除过量的生物素化检测抗体，加入偶联有藻红蛋白的链霉亲和素，并孵育30分钟。 SAV-RPE与生物素化的检测抗体结合，形成四元固相夹心。洗涤去除未结合的SAV-RPE后，用Luminex检测系统分析微珠。通过监测微珠的光谱特性和相关的藻红蛋白（RPE）荧光的量，可以测定一种或多种蛋白质的浓度。Luminex技术与以下Luminex分析仪兼容：
•MAGPIX系统 — 经济实惠、高效、紧凑设计
•Luminex100/200系统 — 多功能、高效、广泛用于多重检测
•FLEXMAP 3D系统 — 高通量（同时分析高达500个）和自动化兼容
以下是一些可能的原因和解决方案：
标准品或样品移液或后续步骤中出错。始终以相同的顺序将溶液快速分配到孔中。分配时，请勿触摸每个微孔上的移液器吸头。使用经过校准的移液器和与该移液器匹配的吸头。检查移液器吸头是否泄漏。
重复使用吸头进行多种样品或不同试剂的移液。在每一种样品或试剂转移时，使用新的吸头。
孔被移液器吸头或洗涤吸头刮擦。将溶液分配到微孔中和吸出微孔时要小心。
在孵育期间，液体从一个孔转移到另一个孔。调整轨道摇床或检查摇床转速是否正确。小心地撕下粘性封板膜。
分配到微孔中的材料体积不正确。按照方案分配试剂。检查移液器的校准。
用血清、培养基或其他缓冲液稀释标准品。在试剂盒提供的标准品稀释缓冲液中稀释标准品。
样品中存在颗粒或沉淀物。在将溶液用于检测之前，通过离心除去任何颗粒/沉淀物。
微孔脏：微孔内部或底部可见碎屑。 检查微孔并翻转微孔板以除去碎屑。每次洗涤后，用吸水薄纸擦拭板底部。切勿将薄纸插入微孔内。
由于外边缘孔和板中心的孔之间的温度不均匀而产生“边缘效应”。在孵育期间平板密封要严，并且当要在37℃下孵育时，将平板放置在培养箱的中心。
以下是一些可能的原因和解决方案：
标准品原液制备不当。按照小瓶标签上的指示稀释冻干标准品，仅使用标准稀释缓冲液或与您的样品基质最匹配的稀释液。
使用了来自具有不同的分析物或不同批号的不同试剂盒的试剂（冻干标准品、标准稀释液缓冲液等）。切勿使用其他试剂盒中的任何组分。
标准品移液或后续步骤中出错。始终以相同的顺序将溶液快速分配到孔中。分配时，请勿触摸每个微孔上的移液器吸头。使用经过校准的移液器和与该移液器匹配的吸头。
以下是一些可能的原因和解决方案：
检测开始时，试剂不在室温（~25 ± 2°C）条件下。在开始检测之前，使试剂回温至室温。
组分储存不当，例如，未在2-8°C下储存。完全按照方案和标签上的指示储存所有组分。
抗兔IgG HRP或链霉亲和素-HRP工作溶液在用于检测前，已制备超过15分钟。在稀释后15分钟内使用稀释的抗兔IgG HRP或链霉亲和素-HRP。
试剂过期。收到试剂盒后立即查看有效日期，并在有效期前使用该试剂盒。
在不正确的波长上读板。使用TMB底物读取ELISA的正确波长为450nm。
TMB溶液失去活性。确保TMB溶液在分配到板孔中之前是透明的。蓝色和/或存在颗粒物质表明产品被污染。如果发现此问题，请联系技术支持。为避免污染，我们建议将检测所需的量分配到以前未使用过的一次性槽中以便移液。丢弃留在槽中的任何TMB溶液，不要将其放回瓶中。避免TMB溶液接触含有金属离子的物品。 不要用铝箔或铝涂层的Mylar纸封板，因为这可能会在没有HRP的情况下导致显色。
尝试使用ELISA检测未优化过的基质中的分析物。使用其他样品类型时，请联系技术支持。
孔被移液器吸头或洗涤吸头刮擦。将溶液分配到微孔中和吸出微孔时要小心。
显色液或终止液使用不当。仅使用试剂盒中提供的显色液和终止液。
以下是一些可能的原因和解决方案：
抗兔IgG HRP或链霉亲和素-HRP工作溶液的稀释不正确。
-使抗兔IgG HRP或链霉亲和素-HRP（100X）的溶液回温至室温，缓慢吸取，并用实验室薄纸（例如，Kimwipe薄纸）擦拭吸头以除去过量的溶液。仅在所提供的HRP稀释液中进行稀释。
孵化时间过长。遵循方案中规定的孵育时间。
在37°C下孵育。在方案中有说明时，在室温（~25±2°C）下进行孵育。
以下是一些可能的原因和解决方案：
洗涤不充分和/或洗涤后排液不充分。如果含抗兔IgG HRP或链霉亲和素-HRP的残留溶液留在孔中，可能使背景升高。
-根据方案洗涤。验证自动洗板机的功能。在每个洗涤步骤结束时，将培养板倒置于工作台面上的吸水薄纸上，使液体完全排出，必要时用力拍打以除去残留的液体。
显色剂在使用前暴露于光线下，导致呈现出蓝色。
-将显色剂保存在其小瓶中，直到您准备好将其分配到板中，然后将其倒入容器中以防止设备污染小瓶。请勿用箔封微孔板。
孵育时间过长或孵育温度过高。
-减少孵育时间和/或降低孵育温度。
移液器、配液容器或带有抗兔IgG HRP或链霉亲和素-HRP的底物溶液的污染。
请勿使用在加入前就呈现蓝色的显色剂。换一瓶新的显色剂。
我们的客户使用种类多样的读板仪。其中一些不能读取高于2 AUFS（满量程的吸光度单位）的吸光度，而其他一些不能读取超过3 AUFS。如果您在室温下孵育30分钟后无法读取1或2 A450值，则可以缩短孵育时间。例如，一些客户发现20分钟是理想的孵育时间，因为他们实验室的环境温度高于~72°F（22°C）。在这种情况下，较高的温度会提高HRP驱动的ELISA反应速度。相反，如果您获得的A450值不够高，则可以相应地增加孵育时间。
ELISA标准曲线不理想有2个主要原因。首先，可能没有遵循推荐的试剂盒标准品的溶解方法。应使用试剂盒中提供的标准稀释剂，根据标签上的指示复溶标准品。请勿使用其他稀释液。然后旋转小瓶或轻轻混匀，然后在室温下静置10分钟以确保完全溶解。这一浓缩的标准品溶液应在复溶后1小时内使用。此外，根据产品手册中提供的说明，在标准品稀释液中制备稀释液之前，应再次轻轻混匀。除非产品手册中另有说明，否则剩余标准品通常可以分装后冷冻储存。
标准曲线不理想的第二个常见原因是HRP偶联物未被正确稀释。100X HRP偶联物溶液含有50％甘油，因此非常粘稠并且难以准确地移液。以下是我们建议解决此问题的方法：首先，让HRP偶联物小瓶达到室温。然后，用干净的移液器吸头轻轻搅拌，以确保均匀。仅使用试剂盒中提供的独立的HRP偶联稀释液对其进行稀释，并按照手册中提供的稀释说明进行操作。
稀释HRP偶联物的关键是确保正确移液。您应该测试您的移液器是否准确地吸出并分配所需的偶联物 - 甘油混合物的体积。如果可能，应该校准此移液器，确保其准确可靠。当吸入粘稠的偶联物溶液时，可能需要5-10秒才能使所需的量进入移液器吸头。在将偶联物转移到合适的HRP稀释液之前，用实验室薄纸（例如，Kimwipes薄纸）擦拭移液器吸头，确保其外部干燥，注意吸头内的内容物不能被薄纸吸收。将偶联物吸移到稀释液中，然后通过上下吹打几次来冲洗吸头。需将最后一滴偶联物移出吸头，这一点尤为重要。接下来，密封容器，并通过轻轻摇晃或上下翻转将其完全混匀。这一步至关重要，因为甘油会将偶联物快速带到试管底部。如果稀释的偶联物未充分混匀，则HRP偶联物的浓度将不是所要求的浓度。
将HRP偶联物稀释并轻轻但完全混匀后，在15分钟内使用。请记住，HRP偶联物稀释液是HRP偶联物唯一可接受的稀释液。不能保存稀释的HRP偶联物，因为HRP活性不稳定，因此不应储存和重复使用。
我们的抗体配对试剂盒包括配对的、预滴定和完全优化的包被和检测抗体，3瓶重组蛋白标准品，链霉亲和素-HRP偶联浓缩物和批次特定的技术数据表。但不含缓冲液。运行抗体对ELISA所需的缓冲液试剂盒必须单独购买（货号CNB0011）。请注意，货号CNB0011中提供的5X检测缓冲液用于封闭您的ELISA微孔板，并作为标准品和样品的稀释液。
不可以。与这些试剂盒中提供的完全相同的链霉亲和素-HRP偶联物不作为单独产品提供。然而，它来自我们确实可以销售的ELISA级链霉亲和素-HRP（货号SNN2004）。请记住，每批ELISA或抗体对试剂盒中提供的链霉亲和素-HRP也因批次而有所不同。所以，如果你使用货号SNN2004或其他来源的链霉亲和素-HRP，您必须确定哪种稀释度的SNN2004最适合您。
是的，可以使用。传统1X RIPA缓冲液的组分与我们的细胞提取缓冲液（货号FNN0011）非常相似。货号FNN0011经常用于制备裂解物，以与我们的ELISA和Luminex试剂盒一起进行测试。我们还使用NP-40细胞提取缓冲液（货号FNN0021）。我们建议将货号FNN0011制成的裂解物稀释至少10倍，以便在将样品加入ELISA或Luminex检测之前将SDS浓度降低至≤0.01％（v / v）。
我们的磷酸特异性ELISA试剂盒具有几个优点，包括易用性和较高的灵敏度。磷酸特异性ELISA试剂盒的灵敏度通常比蛋白质免疫印迹高2-10倍，因此它们特别适用于检测“低表达”蛋白质或小样品量。此外，通过使用试剂盒中提供的重组标准品，磷酸特异性ELISA可在无需进行光密度测定的情况下提供量化的结果。
为了评价磷酸化水平，我们报告了蛋白质磷酸化的比较水平，以“磷蛋白单位/pg或ng总蛋白”为单位。总ELISA试剂盒量化每个样品的蛋白质质量，磷酸特异性ELISA试剂盒以单位量化该蛋白质的磷酸化水平。然后即可确定各种样品的磷酸化水平（例如，以“单位/ pg”为单位）是相似还是不同。
示例：测试两个样品的总CREB和CREB [pS133]
样品1的结果：总ELISA检测（KHO0231）显示样品中的CREB为100pg / mL。磷酸特异性ELISA（KHO0241）显示CREB [pS133]为50单位/ mL。在此样品中，CREB在丝氨酸133处被磷酸化至（50单位/ mL）/（100pg / mL）= 0.5单位/ pg总CREB的水平。
样品2的结果：总ELISA检测显示样品中的CREB为95 pg / mL。磷酸特异性ELISA显示CREB [pS133]为5单位/ mL。在此样品中，CREB在丝氨酸133处被磷酸化至（5单位/ mL）/（95 pg / mL）= 0.053单位/ pg总CREB的水平。当您将样品1与样品2进行比较时，您会看到CREB1在丝氨酸133处的磷酸化水平差异为10倍，即使总CREB蛋白几乎没有变化。
我们的总ELISA的结果以pg / mL表示，或者有时以ng / mL表示。该测量通常以质量单位给出，因为绘制标准曲线时使用的是已知质量的标准品。磷酸特异性ELISA的结果以“单位”表示，我们没有将其与特定的蛋白质质量相关联。我们使用单位，是因为在磷蛋白标准品的特定制剂中，难以精确地知道特定磷酸化反应的效率，因此难以精确地知道磷酸化蛋白与未磷酸化蛋白的比例。磷酸化单位对于每种磷酸特异性ELISA都是不同的，并且在每个试剂盒随附的产品手册中有描述。
例如，典型的单位描述是“1单位=源自300pg自动磷酸化FAK蛋白的FAK [pY397]的量”。由于不能保证我们的标准制剂中的FAK是100％磷酸化的，因此我们不会声明这相当于300 pg的磷酸化FAK。相反，我们验证了大批磷酸化蛋白质，并用它来开发我们原始检测的单位定义和标准曲线。我们的蛋白质标准品的后续制剂被归一化为蛋白质的原始批次，以确保我们的单位定义在批次之间保持不变。
两种类型的ELISA试剂盒均可在塑料96孔板的孔内捕获总蛋白，无论其磷酸化状态如何。这通过用“泛抗体”包被孔来完成，这种“泛抗体”不区分蛋白质的磷酸化形式和非磷酸化形式，并且不阻断待研究的磷酸化位点。此外，磷酸特异性ELISA试剂盒可对在一种或多种特异性氨基酸上磷酸化的相同蛋白质进行定量。这一检测不使用第二泛抗体，而是使用特异性识别蛋白质被特异性磷酸化时才存在的表位的抗体（即，抗体具磷酸特异性）。
我们建议使用相同的样品同时运行总ELISA和磷酸特异性ELISA。如果无法做到这一点，请务必尽快用两个试剂盒测试相同的样品。
每个抗体对试剂盒包含捕获（包被）抗体、生物素化检测抗体、重组标准品和链霉亲和素-HRP。其他试剂列于试剂盒中的抗体对手册中，也可单独购买（抗体对缓冲液试剂盒，货号CNB0011；5X检测缓冲液，货号DS98200等）。该手册还提供了一个特定的程序，并说明了遵循特定程序时可以获得的标准曲线的示例。
程序概述总结如下：
1）使用稀释的捕获（包被）抗体将微孔板在2-8℃下包被过夜；冲洗微孔板。
2）在包被的微孔板中孵育标准品或样品；冲洗微孔板。
3）在板中孵育稀释的生物素化检测抗体；冲洗微孔板。
4）将链霉亲和素-HRP在微孔板中孵育15-45分钟；冲洗微孔板。
5）使用TMB底物将微孔板孵育10-60分钟，然后用终止液终止反应。
6）在450nm处读取微孔板。
我们建议测定各种应用的最优缓冲液配方、浓度和孵育时间。
We have 5 different cell and tissue extraction buffers suitable for preparing mouse cell and tissue extracts. These buffers can be used to extract cells and tissues from many other species as well. The exact compositions of all of our buffers are proprietary, but they are similar to those described by many researchers.
Four of these buffers can be used to prepare extracts which can be analyzed with our ELISA and Luminex kits and by Western blotting. Our Cell Extraction Buffer (FNN0011) contains extra phosphatase inhibitors and resembles the RIPA formulation that many people use. Our Tissue Extraction Reagents I (FNN0071) and II (FNN0081) contain different concentrations of NaCl and different surfactants, but are otherwise similar to each other. For those who prefer using an extraction buffer containing the detergent NP-40, we have our NP-40 Lysis Buffer (FNN0021). Finally, we sell a Denaturing Cell Extraction buffer (FNN0091) which contains 3 detergents and a chaotropic agent. Extracts prepared with FNN0091 can be analyzed with our ELISA kits and by Western blotting only. These buffers do not contain protease inhibitors, which the investigator should add right before use.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
Here are possible causes and solutions for this issue:
- Check the protocol settings (make sure you select the correct DD settings).
- Check the level of sheath fluid and empty the waste.
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are possible causes and solutions for this issue:
This usually indicates that the beads have been photobleached. This problem can also be caused by exposing the beads to organic solvents. Unfortunately, the assay will have to be repeated because the beads cannot be restored. The beads must be protected from light and organic solvents.
Alternatively, the instrument may be off in its measurements or you may have a calibration issue. Call the manufacturer for a service appointment.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This indicates that an incorrect buffer was used for the final step. The Wash Solution provided in the kit must be used for washing the beads and the Reading Buffer should be used for resuspending the beads before loading them into the Luminex instrument. The osmolarity of the solution will impact the size of the bead, and any change in the bead size will alter detection by the instrument.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
- Review the instrument settings and make sure they are appropriate for the assay being run (adjustment of needle height, make sure you select the correct bead gates and the correct DD settings).
- Shake the plate before acquisition on the instrument to resuspend the beads.
- Vortex the beads for 30 sec before adding them into the plate.
- Washing: Do not forget to keep the plate for about 2 mins on the Hand-Held Magnetic Plate Washer before emptying the plate.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This pattern is indicative of a sample matrix effect. Here are some suggestions:
- Confirm that the sample has been clarified and is free of debris and free of lipids (5-10 min centrifugation recommended).
- Confirm that there is at least a 1:1 ratio of sample to assay diluent for serum, plasma samples. For cell lysates or tissue homogenates, confirm that the sample has been diluted appropriately in assay buffer to reduce the concentration of detergent in the lysis buffer to ⋜0.01%. For other sample types, further sample optimization may be required.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- It is possible that the levels of your protein of interest fall below the detection limits of the assay. High Sensitivity Multiplex kits are available for most cytokines.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- Sample optimization may be needed: Dilute the sample with an appropriate diluent and re-run.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
To process cell lysates (extract cellular proteins), follow the instructions provided here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017835_PrepareCellLysates_Procartaplex_PI.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We have not specifically tested saliva samples in-house and therefore these instructions are only our recommendation. Saliva contains several proteolytic enzymes. It would be important to centrifuge samples and be sure not to pipet any cellular material or debris into the assay plate. We would suggest adding some anti-protease in the sample (for example, trasylol or aprotinine, 10 to 50 U/mL) to protect the protein from enzyme degradation. You may then centrifuge the samples at 1000 x g at 4 degrees C for 10 mins to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We have not specifically tested oral mucosal transudate samples in-house and therefore these instructions are only our recommendation. Isolate the site around the tooth and insert a piece of periodontal filter paper into the gum pocket around the tooth for 30 seconds. Remove the filter paper and extract 4 times with 50 µL PBS for 5 min each at room temperature. The individual extractions can be combined and analyzed. Dilute 2-fold with Assay Diluent before applying to the assay.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We have not specifically tested cervical secretion samples in-house and therefore these instructions are only our recommendation. Cervical sponges should be placed on ice immediately upon collection. Samples should be stored at -20 degrees C for up to one week and then stored at -80 degrees C until ready for assay. After thawing, sponges should be weighed and placed into Eppendorf tubes, using forceps cleaned with ethanol after each transfer. Add 200 µL of ice-cold extraction buffer (recipe below) to each tube and incubate overnight at 4 degrees C. The sponges and extraction buffer can then be transferred to microcentrifuge tubes with 0.2 µm cellulose acetate filters and centrifuged at 13,000 rpm for 10 min at 4 degrees C. The eluate can then be tested for cytokine expression.
Extraction Buffer
50 mM HEPES, pH 7.5
1 mM Na3VO4
150 mM NaCl
1 mM NaF
1 mM EDTA
0.1% Tween 20
25 mM EGTA
10% glycerol
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We have not specifically tested bronchoalveolar lavage (BAL) samples in-house and therefore these instructions are only our recommendation. The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until you are ready to analyze it. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute 2-fold with Assay Diluent before applying to the plate.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Centrifuge samples at 1,400 rpm for 10 mins at 4 degrees C to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.
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We have not specifically tested synovial fluid samples in-house and therefore these instructions are only our recommendation. Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 min within 30 min of sample collection. The acellular portion of synovial fluid should be stored at -80 degrees C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784-R795.
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Collect spleen, lung, brain, kidney, liver, or heart tissue and treat with or without LPS (100 µg, i.p., for 15 mins, 30 mins, 1 hr, 2 hrs, or 3 hrs). Weigh tissue in a 2 mL microcentrifuge tube. Add 500 µL of Cell Lysis Buffer (Cat. No. EPX-99999-000) per 100 mg of tissue. Add one 5-mm Stainless Steel Bead, then assemble tubes into TissueLyser according to the manufacturer’s recommendations. We recommend using 5-mm Stainless Steel Beads from Qiagen (Cat. No. 69989). Homogenize tissue at 25 Hz for 0.5-3 mins as indicated in the table below. Centrifuge the sample at 16,000 × g for 10 mins at 4 degrees C. Transfer the supernatant to a new microcentrifuge tube. Measure the total protein concentration. Dilute samples to 10 mg protein/mL with 1X PBS. To proceed with ProcartaPlex protocol, add 25 µL of Universal Assay Buffer (Cat. No. EPX-11111-000) to 25 µL of the diluted sample to each sample well.
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Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at -80 degrees C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.
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Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.
If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4 degrees C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.
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Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to clot for 20-30 mins at 20-25°C. Centrifuge at 1,000 x g for 10 mins at 20-25°C and collect the serum fraction. Alternatively, a serum separator tube can be used following the manufacturer's instructions. Use immediately or store aliquots at -80°C. Avoid multiple freeze-thaw cycles.
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ProcartaPlex multiplex assays, which are based on Luminex xMAP technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, ProcartaPlex multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of ProcartaPlex multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:
Specificity-each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
Sensitivity-each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
Precision/accuracy-multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests
ProcartaPlex multiplex assays are regularly tested against the matching ELISAs. Therefore, you can switch easily from ProcartaPlex assays to ELISA and vice versa with reliable results. Most of our ProcartaPlex assays use the same antibody pairs as our traditional plate-based ELISAs, resulting in high correlation (R2 > 0.9) between the two assays.
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ELISA is a simple and powerful way to quantify individual proteins specifically in complex samples. The selectivity of ELISA is achieved through the use of qualified single- or double-antibody sandwich technology, and accurate quantitation is achieved through the use of calibrated standards. ELISAs can detect low-level proteins and can be performed in a 96-well format with only 60 minutes of hands-on time. In addition, the results obtained with ELISAs are generally very reproducible. While ELISA has been established as a standard method of protein analysis, multiplexing methods that enable the measurement of multiple analytes simultaneously in a single sample address a number of specific limitations:
ELISA allows for the measurement of only one analyte at a time in a given sample, limiting investigators' increasing need to measure multiple targets in their research studies.
The low available volume of many samples being studied may limit the number of times analyses can be conducted. This is especially true in small animal research, in pediatric testing, and in microplate assays providing limited sample volumes. The ability to assay multiple analytes in a single small-volume sample enables more effective use of each sample.
Difficulties in data interpretation can arise when comparing analyte levels measured by multiple ELISAs, each assay having been performed with different sample aliquots and each susceptible to systematic errors leading to decreased precision and accuracy.
Many analytes require assays with broad dynamic ranges to avoid repeat testing or out-of-range values. Multiplex assays can be designed to have large dynamic ranges for all of the analytes, or ranges tailored to various expected analyte concentrations.
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Luminex xMAP technology is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities. Each dyed bead is given a unique number, known as a “bead region”, allowing the differentiation of beads. For ProcartaPlex multiplex immunoassay kits, individual bead sets are then coated with a capture antibody qualified for one specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well assay to detect and quantify multiple targets simultaneously, using one of the Luminex instruments for analysis.
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The Luminex assay is a bead-based immunoassay that uses beads of defined spectral properties conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration and test samples) into the wells of a microplate. The target protein binds to the capture antibodies over the course of a 2 hr incubation. After incubation on a shaker, the beads are washed by putting the 96-well plate on a flat magnet for 30 seconds, after which the fluid is discarded by flicking the wells or by using an automated plate washer. The magnet is removed, and the beads are resuspended in the detection antibody. Another incubation and wash are followed by the addition of streptavidin–R-phycoerythrin (SAPE). The beads are then washed and are ready to analyze. The Luminex technology is compatible with the following Luminex analyzers:
MAGPIX System-affordable, efficient, and compact
Luminex 200 System-versatile, efficient, and widely used in multiplexing
FLEXMAP 3D System-high throughput (up to 500 simultaneous assays) and automation compatible
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Here are possible causes and solutions:
Errors in pipetting the standards or samples or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device. Check for any leaks in the pipette tip.
Repetitive use of tips for several samples or different reagents. Use fresh tips for each sample or reagent transfer.
Wells have been scratched with the pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Liquid transferred from well to well during incubations. Adjust the orbital shaker or check for correct rotator rpm. Peel the adhesive plate cover off carefully.
Incorrect volumes of materials dispensed into the microwells. Follow the protocol for dispensing volumes of reagents. Check calibration of the pipettes.
Standard diluted with the serum, culture medium, or other buffer. Dilute the standard with the standard diluent buffer provided in the kit.
Particulates or precipitates present in the samples. Remove any particulates/precipitates by centrifugation prior to dispensing into the assay.
Dirty microwells: visible debris within or on bottom of microwells. Inspect the microwells and invert the plate to remove debris. Wipe the bottom of the plate with an absorbent tissue after each wash step. Never insert tissue into the microwells.
“Edge effect” due to uneven temperature between the outer-edge wells and the wells in the center of the plate. Seal the plate completely with a cover during incubations, and place the plate in the center of the incubator when 37 degrees C incubation is indicated.
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Here are possible causes and solutions:
Improper preparation of standard stock solution.Dilute the lyophilized standard as directed on the vial label, only with the standard diluent buffer or a diluent that most closely matches the matrix of your sample.
Reagents (lyophilized standard, standard diluent buffer, etc.) from different kits, with either different analytes or different lot numbers, were substituted. Never substitute any components from another kit.
Errors in pipetting the standard or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tips on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.
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Here are possible causes and solutions:
Reagents not at room temperature (approximately 25 plus or minus 2 degrees C) at start of assay. Allow all reagents to warm to room temperature prior to commencing the assay.
Incorrect storage of components, e.g., not stored at 2-8 degrees C. Store all components exactly as directed in the protocol and on labels.
Anti-rabbit IgG HRP or streptavidin-HRP working solution made more than 15 minutes before use in assay. Use the diluted anti-rabbit IgG HRP or streptavidin-HRP within 15 minutes of dilution.
Expired reagents.Check expiration dates upon receipt of kit and use the kit prior to expiration.
Plate read at incorrect wavelength. The correct wavelength to read ELISAs using the TMB substrate is 450 nm.
TMB solution lost activity. Ensure that the TMB solution is clear before it is dispensed into the plate wells. A blue color and/or the presence of particulate matter indicate that the product is contaminated. Please contact Technical Support if this problem is noted. To avoid contamination, we recommend that the quantity required for an assay be dispensed into a previously unused disposable trough for pipetting. Discard any TMB solution left in the trough and do not put it back in the bottle. Avoid contact between the TMB solution and items containing metal ions. Do not cover your plates with aluminum foil or aluminum-coated Mylar sheets because this can cause color development in the absence of HRP.
Attempt to measure analyte in a matrix for which the ELISA assay is not optimized. Contact Technical Support when using alternative sample types.
Wells have been scratched with pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Incorrect chromogen or stop solution used. Use only the chromogen and stop solution supplied with the kit.
Standard diluent buffer added to all wells rather than the designated wells. Follow the protocol and only add the standard diluent to the designated wells and to the samples where it is required, or to samples producing signals greater than that of the highest standard.
Use of buffer containing azide, which is not compatible with HRP. Avoid the use of azide in the assay.
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Here are possible causes and solutions:
Incorrect dilution of the anti-rabbit IgG HRP or streptavidin-HRP working solution.
Warm the solution of anti-rabbit IgG HRP or streptavidin-HRP (100X) to room temperature, draw it up slowly, and wipe the tip with a laboratory tissue (e.g., Kimwipe tissue) to remove the excess. Dilute only in the HRP diluent provided.
Incubation times extended. Follow incubation times outlined in the protocol.
Incubations performed at 37 degrees C. Perform incubations at room temperature (approximately 25 plus or minus 2 degrees C) when instructed in the protocol.
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Here are possible causes and solutions:
Insufficient washing and/or draining of wells after washing. Residual solution containing anti-rabbit IgG HRP or streptavidin-HRP can elevate the background if left in the well.
Wash according to the protocol. Verify the function of the automated plate washer. At the end of each washing step, invert the plate on absorbent tissue on the countertop and allow it to completely drain, tapping forcefully if necessary to remove residual fluid.
Chromogen exposed to light prior to use, resulting in a blue color.
Keep chromogen in its vial until you are ready to dispense it into the plate, and then pour it into a reservoir to prevent contamination of the vial with equipment. Do not cover the plate with foil.
Incubation time is too long or incubation temperature is too high.
Reduce incubation time and/or temperature.
Contamination of pipette, dispensing reservoir, or substrate solution with anti-rabbit IgG HRP or streptavidin-HRP.
Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain a new vial of chromogen.
Blanks that have been set up improperly.
Follow the protocol when designating blank wells. Blank wells contain only chromogen and stop solution. Subtract blank well results from all other wells.
Incorrect dilution of standard stock solution or standards diluted in serum, culture supernatant, or other.
Follow the protocol instructions regarding dilution of the standard. Dilute standards only in the Standard Diluent Buffer provided in the kit.
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Our customers use a wide variety of plate readers. Some of these can't read absorbances higher than 2 AUFS (Absorbance Units Full Scale), while others can't go beyond 3 AUFS, for example. If you read your ELISA plates after 30 minutes of incubation at room temperature and 1 or 2 A450 values are off-scale, you can shorten the incubation time. For example, some customers find that 20 minutes is the ideal incubation time because the ambient temperature in their lab is higher than approximately 2 degrees F (22 degrees C). In this case, higher temperatures increase the rate of the HRP-driven ELISA. Conversely, if the A450 values you get are not high enough, you can increase the incubation time accordingly.
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There are 2 main causes of poor ELISA standard curves. First, the recommended method for solubilizing the kit standard may not have been followed. The standard should be reconstituted according to the directions indicated on the label, using the standard diluent provided in the kit. No other diluent should be used. The vial should then be swirled or mixed gently and then allowed to sit for 10 minutes at room temperature to ensure complete solubilization. This concentrated standard solution should be used within 1 hour of reconstitution. Also, it should be mixed gently again before preparing the dilutions in standard diluent according to the instructions provided in the product manual. Leftover standard can usually be stored frozen in small aliquots, unless specified otherwise in the product manual.
The second common reason for poor standard curves is that the HRP conjugate was not diluted correctly. The 100X HRP conjugate solution contains 50% glycerol, which makes it very viscous and difficult to pipet accurately. Here is what we suggest to solve this problem: First, let the vial of HRP conjugate come to room temperature. Then, stir it gently with a clean pipet tip to make sure that it is homogeneous. Use only the separate HRP conjugate diluent provided in the kit to dilute it, and follow the dilution instructions provided in the manual.
The key to diluting the HRP conjugate is to make sure that it is pipetted correctly. You should test that your pipettor accurately aspirates and dispenses the volume of the conjugate-glycerol mixture that is required. If possible, this pipettor should be calibrated so it is accurate and reliable. When you aspirate the viscous conjugate solution, it may take 5-10 seconds for the desired amount to enter the pipet tip. Before transferring the conjugate to the appropriate HRP diluent, make sure that the outside of the pipet tip is dry by wiping it with a lab tissue (e.g., Kimwipes tissue), taking care to ensure that the contents inside the tip do not get absorbed by the tissue. Pipet the conjugate into the diluent, and then rinse out the tip by pipetting up and down several times. It is important to get every last bit of conjugate out of the tip. Next, seal the container and mix it gently but thoroughly by rocking it or turning it upside down. This is crucial because the glycerol carries the conjugate quickly down to the bottom of the tube. If the diluted conjugate is not mixed adequately, the concentration of the HRP conjugate will not be what is required.
Once the HRP conjugate is diluted and mixed gently but well, use it within 15 minutes. Remember that the HRP conjugate diluent is the only acceptable diluent for the HRP conjugate. The diluted HRP conjugate should not be saved because the HRP activity is labile, and it should never be stored and reused.
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Our Antibody Pair kits include matched, pre-titered and fully optimized coating and detection antibodies, 3 vials of recombinant protein standard, the streptavidin-HRP conjugate concentrate, and a lot-specific technical data sheet. The buffers are not included. The Buffer Kit needed to run the Antibody Pair ELISA must be purchased separately (Cat. No. CNB0011). Note that the 5X Assay Buffer supplied in Cat. No. CNB0011 is used to block your ELISA plates and as a diluent for the standards and samples.
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No. The exact same streptavidin-HRP conjugate supplied in these kits is not available as a stand-alone product. However, it is derived from our ELISA-grade streptavidin-HRP (Cat. No. SNN2004), which we do sell. Remember that the streptavidin-HRP provided in each lot of ELISA or Antibody Pair kits is also lot-specific. So, if you use Cat. No. SNN2004 or another source of streptavidin-HRP, you will have to determine which dilution of SNN2004 works best for you.
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Yes, you can. The composition of the traditional 1X RIPA buffer is very similar to that of our Cell Extraction Buffer (Cat. No. FNN0011). Cat. No. FNN0011 is frequently used to prepare lysates for testing with our ELISA and Luminex kits. Our NP-40-based Cell Extraction Buffer (Cat. No. FNN0021) is also used. We recommend diluting lysates made with Cat. No. FNN0011 at least 10-fold in order to lower the SDS concentration to less than or equal to 0.01% (v/v) before adding the samples to the ELISA or Luminex assay.
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Our phosphospecific ELISA kits have several advantages, including ease of use and increased sensitivity. Phosphospecific ELISA kits are typically 2-10 times more sensitive than western blots, so they are particularly useful for the detection of “low-expressing” proteins or for small sample sizes. In addition, with the use of the recombinant standards provided in the kit, phosphospecific ELISAs provide quantitative results without having to perform densitometry.
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In order to evaluate phosphorylation levels, we report comparative levels of protein phosphorylation in units of phosphoprotein per pg or ng of total protein. The total ELISA kit quantifies the mass of protein per sample, and the phosphospecific ELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) of various samples are similar or different.
Example: Two samples are tested for total CREB and CREB [pS133]
Sample 1 results: The total assay (KHO0231) shows 100 pg/mL of CREB in the sample. The phosphospecific ELISA (KHO0241) shows 50 units/mL of CREB [pS133]. In this sample, CREB is phosphorylated at serine 133 to the level of (50 units/mL)/(100 pg/mL) = 0.5 units/pg of total CREB.
Sample 2 results: The total assay shows 95 pg/mL of CREB in the sample. The phosphospecific ELISA results show 5 units/mL of CREB [pS133]. In this sample, CREB is phosphorylated at serine 133 to the level of (5 units/mL)/(95 pg/mL) = 0.053 units/pg of total CREB. When you compare sample 1 with sample 2, you see a 10-fold difference in the level of phosphorylation of CREB at serine 133, even though the amount of total CREB protein is nearly unchanged.
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The results of our total ELISAs are given in pg/mL of sample, or sometimes ng/mL. This measurement is always given in mass units because standards of known mass are used to prepare the standard curve. The results of the phosphospecific ELISAs are given in “units”, which we do not relate to a particular mass of protein. We use units because it is difficult to precisely know the efficiency of a particular phosphorylation reaction, and therefore the ratio of phosphorylated to unphosphorylated protein, in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphospecific ELISA and are described within the product manual that accompanies each kit.
For example, a typical unit description would be “1 unit = the amount of FAK [pY397] derived from 300 pg of auto-phosphorylated FAK protein”. Since there is no guarantee that the FAK in our standard preparation is 100% phosphorylated, we refrain from making the statement that this corresponds to 300 pg of phosphorylated FAK. Instead, we validate a large batch of phosphorylated protein and use this to develop our unit definition and standard curve for our original assay. Subsequent preparations of our protein standards are normalized to the original batch of protein to ensure that our unit definitions remain constant from lot to lot.
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Both types of ELISA kits capture total protein, regardless of its phosphorylation state, within the wells of a plastic 96-well plate. This is done by coating the wells with a “pan-antibody” that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein and does not block the phosphorylation site to be studied. In addition, a phosphospecific ELISA kit quantifies the amount of that same protein that is phosphorylated on one or more specific amino acids. Instead of a second pan-antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated specifically (i.e., it is phosphospecific).
We recommend running the total and phosphospecific ELISAs simultaneously with the same samples. If this is not possible, make sure to test the same samples with both kits as soon as possible.
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Each Antibody Pair kit contains capture (coating) antibody, biotinylated detection antibody, recombinant standard, and streptavidin-HRP. Other reagents required are listed in the Antibody Pair manual included with the kit, and can also be purchased separately (Antibody Pair Buffer Kit, Cat. No. CNB0011; 5X Assay Buffer, Cat. No. DS98200; etc.). The manual also provides a specific procedure and illustrates an example of a standard curve that can be obtained when the specific procedure is followed.
A general procedure is summarized here:
1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; wash the plate.
2) Incubate the standards or samples in the coated microplate; wash the plate.
3) Incubate diluted biotinlyated detection antibody in the plate; wash the plate.
4) Incubate streptavidin-HRP in the plate for 15-45 min; wash the plate.
5) Incubate the plate with TMB substrate for 10-60 min, and then stop the reaction with Stop solution.
6) Read the microplate at 450 nm.
We recommend determining optimal buffer formulations, concentrations, and incubation times for individual applications.
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