3种细胞裂解液提取总蛋白在Western blot中的效果分析
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摘要
目的寻找一种适用于培养血管平滑肌细胞总蛋白提取的细胞裂解液配方,以更好地应用于Westernblot分析。方法采用3种常用的细胞裂解液(Ⅰ液,Ⅱ液和Ⅲ液),提取总蛋白后Bradford法测定蛋白含量,SDS-聚丙烯酰胺凝胶电泳分离提取的蛋白,Western blot检测PDGFR-β、Akt、GAPDH和SM22α等不同分子量的蛋白表达情况。结果 3种细胞裂解液得到的蛋白含量差异无统计学意义(P>0.05)。小分子量蛋白(<50 000),细胞裂解液Ⅱ和细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅰ(P<0.05);中等分子量蛋白(50 000～80 000),细胞裂解液Ⅱ和细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅰ,细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅱ(P<0.05);高分子量蛋白(>80 000),细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅰ(P<0.05)。Western blot结果显示:PDGFR-β,细胞裂解液Ⅱ和细胞裂解液Ⅲ所检测到的条带光密度值显著高于细胞裂解液Ⅰ(P<0.05);Akt,细胞裂解液Ⅱ和细胞裂解液Ⅲ所检测到的条带光密度值显著高于细胞裂解液Ⅰ,细胞裂解液Ⅲ所检测到的条带光密度值显著高于细胞裂解液Ⅱ(P<0.05);GAPDH,细胞裂解液Ⅱ所检测到的条带光密度值显著高于细胞裂解液Ⅰ和细胞裂解液Ⅲ(P<0.05);SM22α,细胞裂解液Ⅱ所检测到的条带光密度值显著高于细胞裂解液Ⅰ和细胞裂解液Ⅲ(P<0.05)。结论确定了一种裂解效果更好、能满足对不同分子量蛋白进行Western blot检测的裂解液配方,为以Western blot为主的蛋白检测相关研究提供了一定的参考。
Objective To find a cell lysate suitable for total protein extract from cultured vascular smooth muscle cells for better application in Western blot analysis.Methods Three commonly used cell lysates,solution Ⅰ,solution Ⅱ and solution Ⅲ supplement with protease inhibitors were used to extract total protein from cultured vascular smooth muscle cells.Bradford method was used to determine protein content and SDS-polyacrylamide gel electrophoresis(SDSPAGE)was used to isolate protein.And then,Western blot was used to detect the protein expression of PDGFR-β,Akt,GAPDH and SM22α.Results There was no significant difference in protein concentration among the three cell lysates(P>0.05).The optical density values obtaining from cell lysateⅡ and Ⅲ were higher than that obtained from cell lysateⅠin small molecular weight proteins(<50 000)detection.The optical density values obtaining from cell lysateⅡandⅢ were higher than that obtained from cell lysateⅠ,and those values obtaining from cell lysateⅢ were higher than that obtained from cell lysateⅡin medium molecular weight proteins(50 000-80 000)detection.The optical density values obtaining from cell lysateⅢ were higher than that obtained from cell lysate Ⅰ in high molecular weight protein(>80 000)detection.For Western blot,the band optical density of PDGFR-βdetected by cell lysate Ⅱ and Ⅲ was significantly higher than that obtained from cell lysateⅠ.The band optical density of Akt detected by cell lysate Ⅱ and Ⅲ was significantly higher than that obtained from cell lysateⅠ,and the band optical density of Akt detected from cell lysate Ⅲ was significantly higher than that obtained from cell lysateⅡ.The band optical density of GAPDH detected from cell lysateⅡ was significantly higher than that obtained from cell lysate Ⅰ and Ⅲ,the band optical density of SM22α detected fromcell lysateⅡ was significantly higher than that obtained from cell lysateⅠand Ⅲ(P<0.05).Conclusion An optimal cell lysate was selected to detect the different molecular weight protein from cultured vascular smooth muscle cells,which could provide a suitable protocol for the study of protein function based on Western blot assay.
Objective To find a cell lysate suitable for total protein extract from cultured vascular smooth muscle cells for better application in Western blot analysis.Methods Three commonly used cell lysates,solution Ⅰ,solution Ⅱ and solution Ⅲ supplement with protease inhibitors were used to extract total protein from cultured vascular smooth muscle cells.Bradford method was used to determine protein content and SDS-polyacrylamide gel electrophoresis(SDSPAGE)was used to isolate protein.And then,Western blot was used to detect the protein expression of PDGFR-β,Akt,GAPDH and SM22α.Results There was no significant difference in protein concentration among the three cell lysates(P>0.05).The optical density values obtaining from cell lysateⅡ and Ⅲ were higher than that obtained from cell lysateⅠin small molecular weight proteins(<50 000)detection.The optical density values obtaining from cell lysateⅡandⅢ were higher than that obtained from cell lysateⅠ,and those values obtaining from cell lysateⅢ were higher than that obtained from cell lysateⅡin medium molecular weight proteins(50 000-80 000)detection.The optical density values obtaining from cell lysateⅢ were higher than that obtained from cell lysate Ⅰ in high molecular weight protein(>80 000)detection.For Western blot,the band optical density of PDGFR-βdetected by cell lysate Ⅱ and Ⅲ was significantly higher than that obtained from cell lysateⅠ.The band optical density of Akt detected by cell lysate Ⅱ and Ⅲ was significantly higher than that obtained from cell lysateⅠ,and the band optical density of Akt detected from cell lysate Ⅲ was significantly higher than that obtained from cell lysateⅡ.The band optical density of GAPDH detected from cell lysateⅡ was significantly higher than that obtained from cell lysate Ⅰ and Ⅲ,the band optical density of SM22α detected fromcell lysateⅡ was significantly higher than that obtained from cell lysateⅠand Ⅲ(P<0.05).Conclusion An optimal cell lysate was selected to detect the different molecular weight protein from cultured vascular smooth muscle cells,which could provide a suitable protocol for the study of protein function based on Western blot assay.
引文
[1]Peach M,Marsh N,Miskiewicz EI,et al.Solubilization of proteins:the importance of lysis buffer choice[J].Methods Mol Biol,2015,1312:49-60.
[2]Miao SB,Xie XL,Yin YJ,et al.Accumulation of smooth muscle 22αprotein accelerates senescence of vascular smooth muscle cells via stabilization of p53in vitro and in vivo[J].Arterioscler Thromb Vasc Biol,2017,37(10):1849-1859.
[3]Lv P,Zhang F,Yin YJ,et al.SM22αinhibits lamellipodium formation and migration via Ras-Arp2/3signaling in synthetic VSMCs[J].Am J Physiol Cell Physiol,2016,311(5):C758-767.
[4]Hays TT,Ma B,Zhou N,et al.Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats[J].Aging Cell,2018,17(3):e12748.
[5]Uhrin P,Wang D,Mocan A,et al.Vascular smooth muscle cell proliferation as a therapeutic target.Part 2:natural products inhibiting proliferation[J].Biotechnol Adv,2018,36(6):1608-1621.
[6]陈鹏,赵丽丽,聂茜,等.贴块法培养大鼠主动脉血管平滑肌细胞条件的优化[J].河北医科大学学报,2014,35(10):1220-1222.
[7]陈含冰,张雨晴,刘津尧,等.载脂蛋白Cl表达与血管内膜増生关系的初步研究[J].河北医科大学学报,2017,38(2):125-128.
[8]李晓坤,麻晓婷,聂磊,等.Sirt1转基因小鼠股动脉结扎后血管新生的初步研究[J].河北医科大学学报,2016,37(3):340-342.
[9]Li X,Jung JJ,Nie L,et al.The neuropilin-like protein ESDNregulates insulin signaling and sensitivity[J].Am J Physiol Heart Circ Physiol,2016,310(9):H1184-1193.
[10]Dong LH,Li L,Song Y,et al.TRAF6-Mediated SM22αK21ubiquitination promotes G6PD activation and NADPHproduction,contributing to GSH homeostasis and VSMCsurvival in vitro and in vivo[J].Circ Res,2015,117(8):684-694.
[2]Miao SB,Xie XL,Yin YJ,et al.Accumulation of smooth muscle 22αprotein accelerates senescence of vascular smooth muscle cells via stabilization of p53in vitro and in vivo[J].Arterioscler Thromb Vasc Biol,2017,37(10):1849-1859.
[3]Lv P,Zhang F,Yin YJ,et al.SM22αinhibits lamellipodium formation and migration via Ras-Arp2/3signaling in synthetic VSMCs[J].Am J Physiol Cell Physiol,2016,311(5):C758-767.
[4]Hays TT,Ma B,Zhou N,et al.Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats[J].Aging Cell,2018,17(3):e12748.
[5]Uhrin P,Wang D,Mocan A,et al.Vascular smooth muscle cell proliferation as a therapeutic target.Part 2:natural products inhibiting proliferation[J].Biotechnol Adv,2018,36(6):1608-1621.
[6]陈鹏,赵丽丽,聂茜,等.贴块法培养大鼠主动脉血管平滑肌细胞条件的优化[J].河北医科大学学报,2014,35(10):1220-1222.
[7]陈含冰,张雨晴,刘津尧,等.载脂蛋白Cl表达与血管内膜増生关系的初步研究[J].河北医科大学学报,2017,38(2):125-128.
[8]李晓坤,麻晓婷,聂磊,等.Sirt1转基因小鼠股动脉结扎后血管新生的初步研究[J].河北医科大学学报,2016,37(3):340-342.
[9]Li X,Jung JJ,Nie L,et al.The neuropilin-like protein ESDNregulates insulin signaling and sensitivity[J].Am J Physiol Heart Circ Physiol,2016,310(9):H1184-1193.
[10]Dong LH,Li L,Song Y,et al.TRAF6-Mediated SM22αK21ubiquitination promotes G6PD activation and NADPHproduction,contributing to GSH homeostasis and VSMCsurvival in vitro and in vivo[J].Circ Res,2015,117(8):684-694.
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