20cm与8cm SDS-PAGE检测钙ATP酶同工异构体分子量比较

目的 比较在蛋白质组学中使用的20 cm SDS-PAGE和传统的8 cm SDS-PAGE在细胞膜钙ATP酶(PMCA)同工异构体表观相对分子质量测量的差异.方法 人晶状体上皮细胞(HLE B-3细胞)经培养至90%融合后,收集其膜蛋白.部分样品行20 cm SDS-PAGE,条件为16 mA/gel,然后改为24 mA/gel,共4～5 h.另一部分样品行8 cm SDS-PAGE,条件为200V 45 min.在同一胶上同时电泳已知相对分子质量的蛋白质标记物.分离厚的蛋白转移到PVDF膜上,用针对细胞膜钙ATP酶(PMCA)同工异构体PMCA1、PMCA2和PMCA4的特舁性抗体进行Western印迹检测.通过电泳移行距离与对数相对分子质量的线性关系计算各个同工异构体的表观相对分子质量.结果 从电泳条带的形态上看,8 cm胶中蛋白条带较细而浓集,而20 cm胶中较宽而扩散.在8 cm胶208 000和126 000条带之间的距离为6.1 mm,而在20 cm胶此距离为32.8 mm.在8 cm胶126 000和97 000条带之间的距离为5.2 mm,而在20 cm胶为20.2mm.20 cm胶中各蛋白条带的移行距离较8 cm胶增加.但是在8 cm胶中蛋白质条带的浓集性较好.在8 cm胶中PMCA 1、2、4的表观相对分子质量分别为153 800、153 500和152 900;在20 cm胶中,则分别为153 1130、125 500和147 400.结论 首次证实20cm和8cm SDS-PAGE均能成功榆测HLE B-3细胞中PMCA 1、2、4的存在,但是检测出的表观相对分子质量有所不同.PMCA 1、2、4的相对分子质量在8 cm胶中分别为153 800,153 500和152 900,而在20 cm胶中分别为153 100,125 500和147 400.PMCA2很可能在20 cm胶长时间的电泳过程中发生了蛋白水解现象.

Detection of molecular weight of PMCA isoform with 20cm SDS-PAGE compared with 8cm SDS-PAGE

Objective To compare the effects between 20 cm SDS-PAGE and 80 cm SDS-PAGE in identifying human lens epithelium B-3 (HLE B-3) cells plasma membrane calcium ATPase (PMCA) isoform's apparent molecular weight(MW). Methods HLE B-3 cells were cultured and membrane protein sample were collected. Part of the sample was electrophoresised with 20 cm gel, 16 mA/gel and then 24 mA/gel for 4-5 h. The same sample was electro phoresised with 8 cm mini gel, 200 V for 45 min. Protein marker of known MW was run with the sample in the same gel. The seperated proteins were transferred to PVDF membrane and Western blot was used to identify the PMCA isoform with specific antibody against PMCA 1,2,4. The apparent MW was calculated in reference to the known protein marker which was electrophoresised in the same gel. Results In 8 cm gel, the distance between 208 000 and 126 000 band was about 6.1 mm,while that of 20 cm gel was 32.8 mm. The distance between 126 000 and 97 000 was about 5.2 mm in 8 cm gel, while that of 20 cm gel was 20.2 mm. The migration distance differences of protein bands in 20 cm gel were significantly larger than those in 8 cm gel. But bands were generally more condensed in 8 cm gel. The apparent MW of PMCA 1,2, 4 was 153 800, 153 500 and 152 900 respectively. In 20 cm gel,the apparent MW of PMCA 1,2, 4 was 153 100,125 500 and 147 400 respectively. Conclusions Both the 20 cm gel and 8 cm gel successfully identify PMCA 1 ,2, 4 in HLE B-3 cells. The apparent MW of PMCA 1,2,4 is 153800, 153 500 and 152 900 respectively in 8 cm gel, and 153 100,125 500 and 147 400 respectively in 20 cmgel. PMCA2 probably has some kind of proteolysis during the long electrophoresis time in 20 cm gel.