双向电泳分离技术的比较和优化

COMPARISON AND OPTIMIZATION OF TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS(2-DE) TECHNIQUE

目的:比较优化人血清蛋白双向电泳(2-DE)分离技术,提高2-DE对血清蛋白的分辨能力。方法:收集健康人群标本,分别进行多次双向电泳,比较高丰度蛋白的去除前后及传统的考马斯亮蓝染色和SYPRO-ruby荧光染色两种不同的染色方法等因素对双向电泳图谱质量的影响。结果:考马斯亮蓝染色和SYPRO-ruby荧光染色相比,血清2-DE图谱检测到平均蛋白质点数分别是(324±25)(n=3)和(785±30)(n=3);去除高丰度蛋白前后比较,未做处理的血清、去除蛋白和IgG血清的2-DE图谱检测到平均蛋白点数分别是(501±25)(n=3)和(813±22)(n=3)。结论:去除高丰度蛋白并结合SYPRO-ruby荧光染色可以大大提高血清低丰度蛋白的解析能力。此方法优化了血清蛋白质2-DE技术,为进一步开展疾病血清蛋白质组研究奠定了基础。

Objective：To compare and optimize a two-dimensional polyacrylamide gel electrophoresis（2-DE） technique,and to enhance the resolution for serum proteomics.Methods：Serum samples were obtained from healthy donors. The effectiveness of different conditions was compared repeatedly according to the final results on 2-DE maps. Depleting high abundance protein,Coomassie Brilliant Blue and SYPRO ruby fluorescent dye staining on the quality of 2-DE maps were compared.Result：The average spots in the Coomassie Brilliant Blue staining gels and SYPRO ruby fluorescent dye staining gels for 2-DE profiles were （324±25）（n=3）and （785±30）（n=3）respectively; and the average spots in the gel for neat serum and high abundance protein depletion serum were （501±25）（n=3）and （813±22）（n=3） separately.Conclusion：Depletion of high-abundance protein combined with SYPRO ruby fluorescent dye staining could enhance low abundance protein detection sensitivity. Optimized 2-DE technique of serum protein can be effectively applied in serum proteomics of diseases.