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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多抗,稀释度更大,特异性相对较差,容易出现多条带。
兔的单克隆抗体和鼠的单克隆抗体在使用上不会有什么区别。
用来很多抗体,许多时候觉得单抗多抗也未必是理论上那样的。单抗做不好的也有,多抗条带唯一且清晰的也有。
而且很多蛋白的抗体未必有那么多的选择。
其次,察看次目的蛋白的存在形式,有没有多聚体形式及变构形式;
最后,查看多家抗体公司的DATA,看看别人的WB做出来的条带的位置。
根据你说的,特异识别多个组织中的同样大小的条带,我觉得很可能就是你的目的蛋白。
单克隆抗体结构相似且均一,有高度特异性
又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体.
抗原有两个基本特性,即抗原性和免疫原性。有抗原性的物质不一定有免疫原性,所以由此引出半抗原和完全全抗原,半抗原必须经过经过一定的改造(偶联蛋白载体BSA,OVA或者HSA等大分子物质)方能成为完全。一般而言完全抗原分子量越大(大于10KDa),结构越复杂引起免疫反应的能力也就越强。
抗体就是能与特异性抗原结合的免疫球蛋白,抗体一般分为多克隆抗体和单克隆抗体,多克隆抗体能与抗原的多个表位结合。本篇主要讲述兔来源的多克隆抗体的生产步骤
多抗一般制备流程:完全抗原的准备→兔子的免疫→ 效价检测和终放→抗体亲和纯化→抗体的浓缩和保存。
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