- Description
- Additional Information
- Readable Documents
- Assay Principle
- Reviews
Key Benefits
- Highly effective and stable chemiluminescent assay for SEAP.
- Extremely sensitive, detect alkaline phosphatase at attogram level.
- Simple and fast assay-add the reagent directly to your experimental samples. Plate can be incubated and read in 5 min.
- Works for cell culture media and tissue lysates.
Additional information
| Kit Size | 100 |
|---|
Secreted alkaline phosphatase (SEAP) is widely used as a reporter to study gene expression. SEAP is secreted into the culture supernatant of the transfected cell lines. SEAP activity in the culture medium reflects changes in intracellular concentrations of SEAP mRNA and protein. The changes of gene expression can be easily assayed using the same cultures by repeated sampling. Cell Technology’s aCellaTM – SEAP detection kit utilizes a Chemiluminescent-based assay technique to detect SEAP reporter gene expression. It provides an extremely sensitive tool to quantitate SEAP and analyze transcriptional and promoter activity in transfected cells.
In the assay, a chemiluminescent substrate was used to detect the Secreted Alkaline Phosphatase in cell culture. The stabilized 1, 2-dioxetane substrate provide high signal to noise ratio, wide dynamic range, rapid results and excellent reproducibility. The SEAP enzyme transfers the phosphoryl residue via a phosphoryl-enzyme intermediate. The intermediate is not stable and decomposed and emits light for detection. The assay can detect alkaline phosphatase at attogram level. The kit provides sample material for 100 assays in a 96-well plate format.
Figure 1. Standard curve of Alkaline Phosphatase (AP). 50 µl serial dilutions of AP (Starting dose 100 mU in tubes) were added to the wells of 96-well luminescent plate. 50 µl of Chemiluminescent substrate was added and the plate was read immediately with a luminometer.
Figure 2. Detection of the functional activity of NF-ĸB/SEAP stable reporter cell line. NF-ĸB/SEAP reporter cells (IML-101) were plated in 96-well plates at 50,000 cells/well. After 24 hours of incubation, cells were stimulated with different doses of TNFα for another 24 hours. 50 µl culture supernatant was added to the wells of 96-well white opaque luminescent plate. 50 µl of chemiluminescent substrate was added and the plate was read immediately with a luminometer.
Figure 3. Inhibition of TNFα activity by a quinazoline TNFα inhibitor (EMD Millipore Cat# 545380-34-5) with NF-ĸB/SEAP stable reporter cell line. NF-ĸB/SEAP reporter cells (IML-101) were plated in 96-well plates at 50,000 cells/well. After 24 hours, 10 ng/ml of TNFα and different dose of TNFα inhibitors were added into each wells. Cells were incubated at 37°C for 24 hours. 50 µl culture supernatant was added to the wells of 96-well white opaque luminescent plate. Then 50 µl of Chemiluminescent substrate was added and the plate was read immediately with a luminometer.
| Document Title |
| aCellaSEAPProtocol |
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免疫组织化学技术按照标记物的种类可分为免疫荧光法、免疫酶法、免疫铁蛋白法、免疫金法及放射免疫自显影法等。免疫荧光细胞化学技术将已知抗体标上荧光素,以此作为探针检查细胞或组织内的相应抗原,在荧光显微镜下观察.当抗原抗体复合物中的荧光素受激发光的照射后会发出一定波长的荧光,从而可以确定组织中的抗原定位或定量.免疫酶细胞化学技术是目前免疫组织化学研究中最常用的技术.基本原理是先以酶标记的抗体与组织或细胞作用，然后加入酶的底物，生成有色的不溶性产物或具有一定电子密度的颗粒，通过光镜或电镜，对细胞或组织内的相应抗原进行定位或定性研究.免疫胶体金技术就是用胶体金标记一抗，二抗或其他的能特异性的结合免疫球蛋白的分子(如葡萄球菌A蛋白)等作为探针对组织或细胞内的抗原进行定性，定位或定量研究.由于胶体金的电子密度高，多用于免疫电镜的单标记或多标记的定位研究。近年来，随着免疫组织化学技术的发展和各种特异性抗体的出现，使许多疑难肿瘤得到了明确诊断。在常规肿瘤病理诊断中，5%-10%的病例单靠H.E.染色难以作出明确的形态学诊断。尤其是免疫组化在肿瘤诊断和鉴别诊断中的实用价值受到了普遍的认可，其在低分化或未分化肿瘤的鉴别诊断时，准确率可达50%-75%。
2．放射免疫检测放射免疫检测技术是目前灵敏度最高的检测技术,利用放射性同素标记抗原（或抗体）,与相应抗体（或抗原）结合后,通过测定抗原抗体结合物的放射性检测结果.放射性同位素具有pg级的灵敏度,且利用反复曝光的方法可对痕量物质进行定量检测.但放射性同位素对人体的损伤也限制了该方法的使用.
3．酶联免疫吸附试验（ELISA）酶联免疫检测是目前应用最广泛的免疫检测方法.该方法是将二抗标记上酶,抗原抗体反应的特异性与酶催化底物的作用结合起来,根据酶作用底物后的显色颜色变化来判断试验结果,其敏感度可达ng水平.常见用于标记的酶有辣根过氧化物酶(HRP)、碱性磷酸酶（AP）等.由于酶联免疫法无需特殊的仪器,检测简单,因此被广泛应用于疾病检测.常用的方法有间接法、夹心法以及BAS－ELISA.间接法是先将待测的蛋白抱被在孔板内,然后依次加入一抗、标记了酶的二抗和底物显色,通过仪器（例如酶标仪）定量检测抗原.这种方法操作简单但由于高背景而特异性较差.目前已逐渐被夹心法取代.夹心法利用二种一抗对目标抗原进行捕获和固定,在确保灵敏度的同时大大提高了反应的特异性.近年来,抗原的定量检测技术也不断推陈出新.近年来,在夹心法ELISA的基础上,开发了多抗原检测试剂盒,能同时检测微量液相样本中多个抗原含量.这项技术的应用大大缩短了诊断的时间,提高诊断的可靠性和及时性.
4．免疫金胶体技术胶体金技术经过30多年的发展到现在已日趋成熟,该方法是将二抗标记上胶体金颗粒,利用抗原抗体间的特异性反应,最终将胶体金标记的二抗吸附于渗滤膜上,此方法简单,快速,广泛应用于临床筛查.
1. 免疫荧光细胞化学技术把已知抗体标上荧光素,以此作为探针检查细胞或组织内的相应抗原,在荧光显微镜下观察.当抗原抗体复合物中的荧光素受激发光的照射后会发出一定波长的荧光,从而可以确定组织中的抗原定位或定量。2. 免疫酶细胞化学技术是目前免疫组织化学研究中最常用的技术．基本原理是先以酶标记的抗体与组织或细胞作用，然后加入酶的底物，生成有色的不溶性产物或具有一定电子密度的颗粒，通过光镜或电镜，对细胞或组织内的相应抗原进行定位或定性研究。3.免疫胶体金技术就是用胶体金标记一抗，二抗或其他的能特异性的结合免疫球蛋白的分子（如葡萄球菌A蛋白）等作为探针对组织或细胞内的抗原进行定性，定位或定量研究．由于胶体金的电子密度高，多用于免疫电镜的单标记或多标记的定位研究。
做荧光抗体试验时，定量测定荧光素的含量用到的是哪个波长？
荧光素是具有光致荧光特性的染料，荧光染料种类很多。目前常用于标记抗体的荧光素有以下几种：异硫氰酸荧光素，四乙基罗丹明，四甲基异硫氰酸罗丹明，酶作用后产生荧光的物质。
在SPSS软件统计结果中，不管是回归分析还是其它分析，都会看到“SIG”，SIG=significance，意为“显著性”，后面的值就是统计出的P值，如果P值0.01<P<0.05,则为差异显著，如果P<0.01,则差异极显著。
F值是方差检验量，是整个模型的整体检验，看你拟合的方程有没有意义
t值是对每一个自变量（logistic回归）的逐个检验，看它的beta值β即回归系数有没有意义
T的数值表示的是对回归参数的显著性检验值，它的绝对值大于等于ta/2(n-k)（这个值表示的是根据你的置信水平，自由度得出的数值）时，就拒绝原假设，即认为在其他解释变量不变的情况下，解释变量X对被解释变量Y的影响是显著的。
F的值是回归方程的显著性检验，表示的是模型中被解释变量与所有解释变量之间的线性关系在总体上是否显著做出推断。若F>Fa(k-1,n-k),则拒绝原假设，即认为列入模型的各个解释变量联合起来对被解释变量有显著影响，反之，则无显著影响。
