The Phosphoprotein Enrichment Kit provides an effective affinity-based procedure for isolating phosphorylated proteins from mammalian cells and tissues. Each kit includes a complete set of buffers along with six high-capacity columns for enrichment of both cytosolic and membrane-bound phosphoproteins, regardless of the amino acid modified—including serine, tyrosine, or threonine.
The Phosphoprotein Enrichment Kit provides an effective affinity-based procedure for isolating phosphorylated proteins from mammalian cells and tissues. Each kit includes a complete set of buffers along with six high-capacity columns for enrichment of both cytosolic and membrane-bound phosphoproteins, regardless of the amino acid modified—including serine, tyrosine, or threonine.
Our enrichment procedure offers a number of advantages:
- The procedure is fast; the average cell-to-sample purification time is less than 2 hours.
- It is also straightforward, consisting of four main steps: adding Extraction/Loading Buffer to the cell or tissue pellet to extract total cellular protein, loading the extract on an affinity column, washing, and finally eluting the bound phosphoprotein with a detergent-free Elution Buffer.
- A single buffer—Extraction/Loading Buffer—is used for both the protein-extraction and affinity-column steps, making buffer exchange unnecessary. This saves time and prevents sample loss.
- The procedure is nondenaturing, so phosphoproteins remain folded throughout the process, even during the extraction and elution steps.
Each column has a maximum binding capacity of ~4 mg of phosphorylated protein.
Highly selective enrichment of phosphoproteins
The Phosphoprotein Enrichment Kit may be used with any mammalian cell type. Cell lines tested include NIH 3T3, HEK 293, HeLa, Cos-7, and Jurkat. The enrichment procedure is highly efficient, as demonstrated by Western blotting analyses. Using a colorimetric phosphate detection method, we found the majority of the phosphoprotein in the eluate; negligible traces were detected in the wash fraction.
Phosphoprotein Affinity Columns yield a concentrated solution of phosphoprotein that can be analyzed by several different methods, including mass spectrometry and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
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试剂盒里有详细的说明书，告诉你样品需要多少量，每个试剂需要加入多少量，和详细的实验步骤，一般买来就可以用，不用人教。
所以你问一个样需要多少量是没法回答的，测定过程是要加很多种试剂的。
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
EliKine™ 人 肝细胞生长因子 ELISA定量试剂盒
EliKine™ 人 干扰素-α ELISA定量试剂盒
EliKine™ 人 干扰素-γ ELISA定量试剂盒
EliKine™ 人 白介素-1α ELISA定量试剂盒
EliKine™ 小鼠 白介素-22 ELISA定量试剂盒
EliKine™ 小鼠 转化生长因子-β1 ELISA定量试剂盒
EliKine™ 小鼠 肿瘤坏死因子-α ELISA定量试剂盒
EliKine™ 小鼠 血管内皮生长因子 ELISA定量试剂盒
EliKine™ 大鼠 干扰素-γ ELISA定量试剂盒
EliKine™ 大鼠 白介素-1β ELISA定量试剂盒
EliKine™ 人 促甲状腺激素 ELISA定量试剂盒
只要引物设计的可以在基因组上做，那么理论上是没问题的！
只需要用RT-PCR荧光定量试剂盒里面的第二个，专门用来做Real-timePCR的那些试剂就可以了！
