Peptide Synthesis and Purification Core Facility

仪器仪表。该设施拥有三个蛋白质技术自动肽 合成器和一个Advanced ChemTech（ACT 90）肽合成器。而蛋白质 技术肽合成器可以在较短的持续时间内提供少量的肽（以mg量计），这是由于在氨基酸树脂上添加了十倍过量的引入氨基酸，ACT可以提供10 g量的肽，但该过程很慢，因为在合成的每个阶段确定反应完成后每天添加一种氨基酸。 由于ACT 90中使用的起始树脂较多，我们避免在每个阶段添加超过所需过量的氨基酸衍生物，以节省氨基酸衍生物的成本。对于肽的纯化，核心设施拥有三个Beckman分析型HPLC和两个制备型HPLC单元（Beckman和Varian）。除此之外，核心设施还使用连接至Michel Muller中压液相色谱（MPLC）玻璃柱的BioRad FPLC系统。这些色谱柱具有不同的尺寸，最小的（分析型）含有12 g固定相（C-4或C-18硅胶），最大的色谱柱含有800 g“自制”反相固定相（C-4和C-18反相硅胶）。我们通常用不同链长的脂肪酸烷基化硅胶。我们一次生产2公斤到4公斤的固定相，用于自制的色谱柱。这提供了更高的能力，以净化大 大量的合成肽。这些柱也用于纯化重组蛋白。典型程序将在方法章节中描述。

Instrumentation. The facility possesses three Protein Technology automatic peptide synthesizers and one Advanced ChemTech (ACT 90) peptide synthesizer. While the Protein Technology peptide synthesizers can provide small amounts of the peptides (in mg quantities) in a shorter duration due to the addition of ten times excess of incoming amino acids on to the amino acid-resin, the ACT can provide 10g quantities of peptides but the process is slow since one amino acid is added per day after the completion of reaction is ascertained at each stage of the synthesis. Since the starting resin used in ACT 90 is more, we avoid adding more than the required excess of amino acid derivatives at each stage in order to save costs on amino acid derivatives. For the purification of peptides the core facility possesses three Beckman analytical HPLC and two preparative HPLC units (Beckman and Varian). In addition to this, the core facility utilizes BioRad FPLC systems that are connected to Michel Muller Medium Pressure Liquid Chromatography (MPLC) glass columns. These columns are of various sizes with the smallest (analytical) containing 12g of the stationary phase (C-4 or C-18 silica gel) and the biggest column containing 800g of "home made" reverse phase stationary phase (C-4 and C-18 Reversed Phase silica gels). We routinely alkylate Silica Gel with various chain-lengths of fatty acids. At a time we produce 2 kg to 4kg of the stationary phase for home made columns. This provides a higher capacity to purify large quantities of synthetic peptides. These columns are also used to purify recombinant proteins. A typical procedure will be described in the Methods Section.