Immobilization Of SsDNA And Its Interaction With Protein

As one of the most important research areas in the post-genome era,proteomics is receiving increasing attention.And the research of low abundance proteins becomes one of the bottlenecks of proteomics currently.A new technology based on the structural diversity of peptide library is used to equilibrate different abundant proteins through equal adsorption of high and low abundance proteins,which is in favor of the research of low abundant proteins. At the same time,oligonucleotide library has also been confirmed with structure polymorphism.Therefore,we hope to establish a technology to equilibrate high and low abundance proteins based on the structure polymorphism of oligonucleotide.To achieve this goal,the effect of surface modification on the characteristics of cross-linked agarose gel,the best approcches of immobilization of ssDNA as well as the influence factors of the interaction between ssDNA and protein have been studied in this paper.The results showed that:(1) 70%of the hydroxyl groups on the gel could be activated by CDI,the activated density could reach 4.4mmol/g dry gel,and in a certain range the activated density was proportional to the amount of CDI.After connected with other molecules,the gel showed an improved anti-pressure ability,increased average diameter,smaller pores and increased solid percentage content,due to the weakening of hydrogen bond inside the beads and between the beads and water molecules caused by introduction of nitrogen or phenyl.All the changes mentioned above were related to the modification density,higher modification density resulted in greater changes in the characteristics of the gel.(2)The directly immobilization of ssDNA on polysaccharide cannot guarantee the effective conformation of ssDNA so this material is not suitable in this research;high-density immobilization will lead the formation of hydrogen bonds between ssDNA-immobilized,and it is not conducive to achieve a specific space conformation of ssDNA;the ssDNA indirectly immobilized through avidin-biotin interaction on Sepharose CL 4B has the adsorptive ability for protein,because the decrease of the formation of hydrogen bond between amino groups in nucleotide and hydroxyl groups in Sepharose CL 4B make ssDNA create its specific space conformation correctly.(3)For ssDNA,some conditions with the ability to create its space conformation such as the denaturalization-renaturation mode and the buffer system can affect its the relationship with protein;denaturalization under 80℃and renaturation slowly in room temperature,using binding buffer with some certain salt are suitable for the interaction between ssDNA and protein while the experimental temperature has little effect on this.(4) We can achieve the aim that both enrich the low concentration protein selectively and equilibrate the high and low abundant proteins with ssDNA which lay a reference for the following experiments.