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查看更多产品信息 PRAS40 (Phospho) [pT246] Mouse ELISA Kit - FAQs (KMO0421)
17 个常见问题解答
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。
所有ELISA试剂盒均以夹心ELISA形式提供，其中捕获抗体已经包被在96孔板上。典型的检测使用生物素化的检测抗体，接着使用链霉亲和素-HRP和HRP底物。大多数试剂盒以单个96孔板的形式提供，有些试剂盒提供2个或5个96孔板。试剂盒通常包含：
•包被捕获抗体的96孔联排板
•蛋白质标准品
•洗涤缓冲液，10x
•ELISA 缓冲液/稀释液，10x
•检测抗体（大多数试剂盒中，生物素化）
•HRP试剂（HRP偶联的二抗或链霉亲和素）（100x）
•底物（通常为TMB）
•终止液
•粘性封板膜
运行内部对照以及空白对照和标准曲线是监测任何ELISA试剂盒性能的绝佳方法。内部对照是用试剂盒测量的含有已知量的分析物的样品。内部对照的一个关键属性，以及它们与标准曲线样品的区别在于，它们会尽可能地复制您实际样品的组分。这一点很重要，因为您的样品可能含有影响分析物检测的成分，而这种影响方式与试剂盒中提供的标准稀释液的影响方式不同。换句话说，内部对照可以让您确定在标准稀释液（标准曲线）和样品基质（内部对照）中检测等量的分析物是否会给出相同的结果。同样重要的是要记住，内部对照可以让您对结果的准确度充满信心。内部对照有助于确保不同检测在每次运行时都能始终如一。
内部对照的来源之一是含有已知量的待测分析物的自然存在的样品（如血清或血浆）。这种阳性对照样品可以原样运行，或者也可以稀释至各种浓度，最好用自然存在的相同类型的阴性样品稀释。如果可以的话，最好运行至少2个内部对照。其中一个分析物的浓度通常在标准曲线的最低点和曲线的中点之间，而另一个的浓度在中点和最高点之间。有些研究人员还会运行一个浓度接近曲线中点的内部对照。
如果您无法获得自然存在的对照品，您可以自己制备。假设您要使用ELISA试剂盒（货号KHC0061）测量人血清样品中的白细胞介素-6（IL-6）。首先，您需要一些合并的正常人类血清作为内部对照的样品基质。该血清的IL-6含量最好是微乎其微（但是已知存在），或者低到无法测量。接下来，您应该使用试剂盒中提供的人类IL-6标准，将已知量的人类IL-6添加到该血清中以建立内部对照。按照产品手册中的说明制备标准曲线后，将一些剩余的浓缩IL-6标准品添加到血清基质中。如上所述，如果您的ELISA微孔板中有足够的孔，您可以制备具有高和低IL-6水平或高、中和低水平的对照。
即使您的样品类型不是血清并且您也不是测量IL-6，也可按照上述通用程序使用您的目标蛋白质和样品基质制备内部对照。 点击此处（https://tools.thermofisher.com/content/sfs/brochures/TR0058-Spike-and-Recovery.pdf）查找加标回收率和稀释度线性分析的有价值的资源。如果您需要更多帮助，请联系技术支持LifeScience-CNTS@thermofisher.com。
我们的ELISA试剂盒可分为多个不同的组（参见蛋白质分析手册（https://www.thermofisher.com/us/en/home/global/forms/protein-handbook-registration.html）第35页的表2），考虑的许多因素包括：靶标蛋白质分类、灵敏度、读出方法或检测靶标蛋白的特异性磷酸化状态的能力。
•标准的比色法ELISA试剂盒使用标准比色读出，具有出色的灵敏度和检测范围。典型灵敏度<10 pg / mL，标准曲线范围约为10-250 pg / mL，但有些试剂盒的范围更广。
•超灵敏ELISA试剂盒使用标准比色读出，但能够检测和分析低至0.5 pg/mL的蛋白质。基于0.5-20 pg/mL的测量范围，这些试剂盒特别适用于高度稀释的样品。
•Chemi ELISA试剂盒使用化学发光检测技术获得高灵敏度（<1 pg/mL），并且灵活性高，测量范围可达0.5至2000 pg / mL。
•磷酸化ELISA试剂盒能够特异性检测关键信号蛋白的磷酸化，且特异性极高，其通常用于补充免疫印迹的结果并提供量化数据。
我们提供完整的ELISA试剂盒、试剂组和抗体对。ELISA试剂盒是完整的即用型试剂盒，包含预包被的检测板、所有缓冲液、捕获和检测抗体。大多数试剂盒以单块板子的格式提供，但有些试剂盒提供2或5块板子。试剂组适用于需要核心试剂盒组件但更喜欢自己制备缓冲液并自己包被板子的研究人员。抗体对是配对的检测和捕获抗体，适用于需要处理大量样品的研究人员。
We are proud to offer an expanding line of ELISA assays to quantify the phosphorylation states of various proteins involved in signal transduction. The total ELISA kit quantifies the mass of protein per sample and the phosphoELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) increase, decrease, or remain constant between various samples.
Our method involves the use of two sandwich ELISA assays. Both assays capture total protein, regardless of phosphorylation state, to the wells of an ELISA plate. This is done by coating the plates 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. One assay then quantifies the total amount of protein present in the sample by using a second "pan" antibody for detection of protein regardless of phosphorylation state. This measurement is given in picograms of protein/mL of sample, or sometimes nanograms of protein/mL. The measurement is given in mass units. This is because standards of known mass are available for use in the standard curve for the total assay.
A second assay quantifies the amount of that same protein that has been phosphorylated at a specific amino acid. 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 at a particular amino acid position. This procedure is extremely similar to an immunoprecipitation reaction in which two different antibodies are used; one to capture a protein from a solution, and one to detect the subset of that protein population that is phosphorylated at a certain site. The ELISA assay has several advantages over immunoprecipitation, including ease of use and increased sensitivity. ELISA is also quantitative. This second assay will detect and quantify only the phosphorylated form of a particular protein. This second measurement is given in "units", which we do not relate to a particular mass of protein. This is done because it is difficult to know precisely the efficiency of a particular phosphorylation reaction, and therefore, the ratio of phosphorylated to nonphosphorylated protein in a particular preparation of phosphoprotein standard. Phosphorylation units will be unique to each phosphoELISA, and are described within the protocol booklet that comes with the assay.
For example, a typical unit description would be "1 unit = the amount of AKT[pS473] derived from 100 pg of AKT phosphorylated by MAPKAP2 and PDK1" as is seen for our AKT[pS473] phosphoELISA assay. Since there is no guarantee that the AKT in our standard prep is 100% phosphorylated, we refrain from making the statement that this corresponds to 100 pg of phosphorylated AKT. 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 insure that our unit definitions remain constant from lot to lot. In order to evaluate phosphorylation levels, we report comparitive levels of phosphorylation of a protein, in units of phosphoprotein per picogram or nanogram of total protein. The total ELISA kit quantifies the mass of protein per sample and the phosphoELISA kit quantifies the phosphorylation level of that protein in units. One can then determine if phosphorylation levels (in units/pg, for instance) increase, decrease, or remain constant between various samples.
Example determination: Two samples tested for CREB [pS133]:
Sample 1 results:
CREB (Total) Human ELISA Kit results show 100 pg/mL CREB in the sample. phosphoELISA CREB [pS133] Human ELISA Kit results show 50 units/mL of CREB phosphorylated at serine 133. For this sample, CREB is phosphorylated at serine 133 to the level of (50 units/mL)/(100 pg/mL) = 0.5 units/pg of CREB protein.
Sample 2 results:
CREB (Total) Human ELISA Kit results show 95 pg/mL CREB in the sample. phosphoELISA CREB [pS133] Human ELISA Kit results show 5 Units/mL of CREB phosphorylated at serine 133. For this sample. CREB is phosphorylated at serine 133 to the level of (5 units/ml)/(95 pg/ml) = 0.05 units/pg of CREB protein.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.
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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All ELISA kits are provided in the sandwich ELISA format with capture antibody already coated onto a 96 well plate. Typical detection uses a biotinylated detection antibody followed by Streptavidin-HRP and HRP substrate. Most kits are available as single 96-well plate kits, some are available as 2- and 5-plate kits. Kits typically contain:
96 Well Strip Plate coated with capture antibody
Standard protein
Wash buffer, 10x
ELISA buffer/Diluent, 10x
Detection antibody (in most kits, biotinylated)
HRP Reagent (either secondary antibody or streptavidin conjugated to HRP)(100x)
Substrate (usually TMB)
Stop solution
Adhesive plate covers
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Running internal controls along with the blanks and the standard curve is an excellent way to monitor the performance of any ELISA kit. Internal controls are samples containing known amounts of the analyte being measured with the kit. A key attribute of internal controls, and what differentiates them from the samples of the standard curve, is that they duplicate the composition of your actual samples as closely as possible. This is important because your samples may contain components that affect detection of the analyte differently than does the Standard Diluent provided in the kit. In other words, internal controls allow you to determine if assaying the same amount of analyte in the Standard Diluent (the standard curve) and in the sample matrix (internal controls) gives the same results. It's also important to remember that internal controls give you confidence in the accuracy of your results. Internal controls help ensure that the assay is performing consistently from assay to assay, every time you run it.
One source of internal controls is naturally occurring samples (such as serum or plasma) containing known amounts of the analyte you want to measure. Such positive control samples can be run as-is, or they can be diluted to various known concentrations, preferably with a naturally occurring negative sample of the same type. It's a good idea to run at least 2 internal controls, if you can. One usually has an analyte concentration between the lowest point on the standard curve and the curve's midpoint, while the other has a concentration between the midpoint and the highest concentration. Some researchers also run an internal control that falls close to the midpoint of the curve.
If you don't have access to naturally occurring controls, you can prepare them yourself. Let's say that you will be measuring interleukin-6 (IL-6) in human serum samples with an ELISA kit, Cat. No. KHC0061. First you will need to obtain some pooled normal human sera, which will be used as the sample matrix for your internal controls. Preferably, the IL-6 content of this serum should be negligible (but known) or too low to measure. Next you should add known amounts of human IL-6 to this serum to create the internal controls, using the human IL-6 standard provided in the kit. After you prepare the standard curve as described in the product manual, add some of the leftover concentrated IL-6 standard to the serum matrix. As described above, you can prepare controls with high and low IL-6 levels, or high, medium, and low, if you have enough wells in your ELISA plate.
Even if your sample type is not serum and you're not measuring IL-6, follow the general procedure outlined above to prepare your internal controls with your protein and sample matrix of interest. A valuable resource describing Spike and Recovery and Linearity of Dilution assays can be found here (http://tools.thermofisher.com/content/sfs/brochures/TR0058-Spike-and-Recovery.pdf). If you need more help, please contact Technical Support at techsupport@thermofisher.com.
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Our ELISA kits can be categorized into several different groups (see Table 2, Page 35 of the Protein Analysis Handbook - http://www.thermofisher.com/us/en/home/global/forms/protein-handbook-registration.html), based on a number of factors: target protein class, sensitivity, readout method, or ability to detect specific phosphorylation states of the target protein.
Standard colorimetric ELISA kits use a standard colorimetric readout and allow excellent sensitivity and detection range. Typical sensitivity is less than 10 pg/mL and standard curve range is around 10-250 pg/mL, although there are some kits with wider ranges.
Ultrasensitive ELISA kits use a standard colorimetric readout but enable detection and analysis of proteins to levels as low as 0.5 pg/mL. With measurement range of 0.5-20 pg/mL, these kits are especially useful with highly diluted samples.
Chemi ELISA kits use chemiluminescence detection for high sensitivity (less than 1 pg/mL) and are highly flexible with a wide measurement range from 0.5 to 2000 pg/mL.
Phospho ELISA kits enable the specific detection of phosphorylation of key signaling proteins with high specificity, and are often used to supplement western blot results and provide quantitative data.
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We offer full ELISA kits, Reagent Sets and Antibody Pairs. ELISA kits are complete, ready-to-use kits with pre-coated plates, all buffers, capture, and detection antibodies included. Most kits are single plate format, but some are available in 2- or 5-plate formats. Reagent Sets are for researchers who need the core kit components but prefer to make their own buffers and coat their own plates. Antibody Pairs are matched pairs of detection and capture antibodies for researchers who need to process large numbers of samples.
Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.