以表面修飾之鑽石粉末做為離子交換材質分析蛋白質及胜肽樣本
Abstract
近年來,離子交換層析技術已經在生物分析和分離領域占有一席之地。 蛋白質或是胜肽都能夠以他們本身不同的等電點被分離。 目前最被廣泛使用的管柱材質是玻璃或塑膠微粒。 不過這兩種材質都有一定的缺點,以玻璃材質為基底的管柱無法在鹼性高的環境運作而塑膠則是無法耐受過高的壓力。
在本篇論文中,經由高濃度氧化酸洗修飾表面的鑽石粉末被證實具有離子交換的功能,而鑽石本身的耐酸鹼特性則成了填充在管柱時最大的優勢。
經過實驗證實,填充表面修飾鑽石的管柱(100*2.1mm 使用約0.6公克的鑽石粉末)蛋白質的乘載量為110~120μg。 可分離的樣本包括蛋白質標準品、尿蛋白以及293T細胞的膜蛋白,還可以搭配SPEED以及SDS-PAGE組成一系列完整分析方法。
相較之下,表面無修飾的鑽石粉末管柱雖然能夠進行相近的分析,但是其缺點是較高的背景值以及清洗還原時需要較多的時間,導致在實驗中的表現較差。
表面修飾鑽石管柱有潛力可以應用在二維層析系統,其能忍受髒污樣本以及純化樣本的能力即可應用在第一維管柱或者是前置管柱。
Recent years, ion exchange technique has already played an important role in bio-molecular separation and analysis. Proteins or peptides can be separated by theirs different pI values. The most widely used ion exchange material is silica and polymer based beads. However, these materials may have some disadvantages. Silica based ion exchange material cannot operate under high pH buffer condition. Polymer based material may be crushed under ultrahigh pressure LC system. Ion exchange ability of surface functionalized diamond (ex. oxidative acid treated) was demonstrated and is applied as an ion exchange column material in this essay. Extreme pH and pressure tolerance becomes common advantages for these diamond columns. In the series of experiments the surface functionalized diamond column (100*4.6mm) has a protein binding capacity of around 110~120μg. Different samples such as standard protein mixture, urine proteins, and 293T membrane proteins can be analyzed by this column. This column can couple with the SPEED technique and analyzed by SDS-PAGE. Surface raw diamond column (none oxidative acid treated) can serve the similar purpose. However, the high background signal level, lower resolution and cost more time at the cleaning step become the disadvantage of using the raw diamond. Oxidative acid treated diamond column potentially can be applied to a 2 dimensional LC system. Due to its tolerance for dirty sample and purification ability, a diamond column can serve as the first-dimension column or pre-column to reduce sample pretreatment time and procedure.
Recent years, ion exchange technique has already played an important role in bio-molecular separation and analysis. Proteins or peptides can be separated by theirs different pI values. The most widely used ion exchange material is silica and polymer based beads. However, these materials may have some disadvantages. Silica based ion exchange material cannot operate under high pH buffer condition. Polymer based material may be crushed under ultrahigh pressure LC system. Ion exchange ability of surface functionalized diamond (ex. oxidative acid treated) was demonstrated and is applied as an ion exchange column material in this essay. Extreme pH and pressure tolerance becomes common advantages for these diamond columns. In the series of experiments the surface functionalized diamond column (100*4.6mm) has a protein binding capacity of around 110~120μg. Different samples such as standard protein mixture, urine proteins, and 293T membrane proteins can be analyzed by this column. This column can couple with the SPEED technique and analyzed by SDS-PAGE. Surface raw diamond column (none oxidative acid treated) can serve the similar purpose. However, the high background signal level, lower resolution and cost more time at the cleaning step become the disadvantage of using the raw diamond. Oxidative acid treated diamond column potentially can be applied to a 2 dimensional LC system. Due to its tolerance for dirty sample and purification ability, a diamond column can serve as the first-dimension column or pre-column to reduce sample pretreatment time and procedure.
Description
Keywords
表面修飾鑽石粉末, 離子交換