Effect of different isolations for nucleic acid and protein complex on affinity of enriched library in SELEX experiment
- VernacularTitle:SELEX实验中核酸-蛋白复合物分离方法对富集库亲和力的影响
- Author:
Chengxiang HU
;
Changguo GU
;
Xudong ZHU
;
Lei LI
;
- Publication Type:Journal Article
- Keywords:
systematic evolution of ligands by exponential enrichment;
aptamer;
enriched library;
affinity
- From:Journal of Third Military Medical University
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To compare the effect of different partition of nucleic acid protein complex on the affinity of enriched library in systematic evolution of ligands by exponential enrichment (SELEX). Methods Two protocols were adopted to select the enriched library to transforming growth factor ? receptor Ⅱ(TGF ? RⅡ). Protocol 1: protein was absorbed on the surface of 96 well plate; then, selection was carried out; the binding nucleotide acids were eluted from the supporter directly. Protocol 2: selection was done in solution; nucleotide acid protein complex was captured in nitrocellulose membrane; the binding nucleotide acids were obtained from membrane. Filter biding assay and gel shift assay were performed to detect the affinity of the enriched ssDNA library from different protocols. Results After 4 rounds of selection, the affinity to TGF ? RⅡ was obviously improved in the enriched library from protocol 2 compared with the initial library, while no such improvement was found in the enriched library from protocol 1. Conclusion In the SELEX experiment, the way of selection in solution, then partition of the binding nucleotide acids in filter is easier to enrich the binding fragment from initial ssDNA random library, compared with the way of fixation of target protein to solid supporter, then selection between the solid phase and liquid phase and elution of the binding nucleotide acids from supporter.
