Regeneration And Preparation Of IMAC Proteinchip

With the development of proteomics in recent years, surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) combining chromatographic separation and protein spectrum can quickly analyze the composition of protein in biological samples. Proteinchip has superiority and bright future for application in many fields such as discovery of disease-related molecules, diagnosis, prevention and drug development, especially screening of cancer markers. However, high price without recycling of proteinchip limits its applications and also bring inconvenience to the medical workers and patients. The used IMAC proteinchips were regenerated by phosphate eluent or borate eluent and the effect of the buffer solution concentration, pH, elution time and the eluent to desorption was inspected. In addition, the adsorption behavior of protein using SiOx film as solid carrier also was examined in this paper. Surface morphology of proteinchip was investigated by scanning electron microscope (SEM) and surface adsorption of regeneration proteinchips were analyzed by SELDI-TOF-MS.The experimental results show that adding phosphate or borate buffer solution to eluent has a good effect on the regeneration of IMAC-Cu proteinchip. With the decreasing of pH value of phosphate elution or borate elution, proteins were easier to be removed from the proteinchip, but its destruction to the chemical structure of the proteinchip also increases. Meanwhile, the longer elution time, the better elution effect of proteinchips, but when the time is too long, the surface of the proteinchip would be terribly destroied. Considering the structure of protein, elution effect and so on, the optimal elution time is 72h.The elution effect of protein on IMAC-Cu proteinchip can be significantly enhanced when Gly or EDTA is added to phosphate or borate eluate. It is more conducive to remove all proteins from the surface of proteinchip in phosphate elute with EDTA as the competition eluant and two kinds of competitive eluant could remove all proteins form proteinchip in borate elute. However, comparing with Gly, EDTA has better effect on adsorption. The IMAC proteinchip can be completely regerenated using 0.02mol/L EDTA as competitive eluen in the phosphate eluent of pH=5.0 eluting for 72h.The better elution effect can be obtained using phosphate and borate elute succesively when several kinds of solutions are used to regerenate proteinchips. Following by the order of P-EDTA elute and N-EDTA elute treating 36h separately, the adsorbed proteinon the surface of the protein-chip can be completely removed and the regenerated proteinchip can adsorpt more proteins.Textured silicon oxide as solid carrier is conducive for the metal ion chelater to keep more polar atoms after aldehyding its surface and enhance the protein adsorption capacity. Compared with other surface carriers, the prepared proteinchip using textured surface as solid carrier has larger surface area which causes higher and more types of adsorption protein. In addition, a seiral of steps such as ultrasound and hydroxylation treating the used proteinchip may break the original chemical chain on the chemical treat spot and cause re-growth new chemical chain which can absorb a certain amount of protein.