Detection of molecular weight of PMCA isoform with 20 cm SDS PAGE electrophoresis: compared with 8 cm SDS PAGE.
OBJECTIVE
To compare the effect of 20 cm SDS-PAGE electrophoresis, which is most widely used in proteomic research, in identifying human lens epithelium B3 (HLE B3) cells plasma membrane calcium ATPase (PMCA) isoform's apparent molecular weight (MW), with that of 8 cm SDS-PAGE electrophoresis.


METHODS
HLE B-3 cells were cultured and membrane protein sample was collected. Part of the sample is electrophoresised with 20 cm gel, 16 mA/gel for 1.5 hrs and then 24 mA/gel for 4-5 hrs. The same sample is electrophoresised with 8cm mini gel, 200V for 2 hrs. Protein marker of known MW was run with the sample in the same gel. The resulting separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane and Western blot were 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 that was electrophoresised in the same gel.


RESULTS
In 8 cm gel the distance between 208 kDa and 126 kDa band was about 6.1 mm, while that of 20 cm gel was 32.8 mm. The distance between 126 kDa and 97 kDa was about 5.2 mm, while that of 20 cm gel was 20.2 mm. The migration distance differences of protein bands were significantly much longer in 20 cm gel than in 8 cm gel (P < 0.005). But the bands were generally more condensed in 8 cm gel. The apparent MW of PMCA1, 2, 4 were 153.8, 153.5 and 152.9 kDa respectively. In the 20 cm gel, the apparent MW for PMCA1, 2, 4 was 153.1, 125.5 and 147.4 kDa respectively.


CONCLUSIONS
Both the 20 cm gel and 8 cm gel successful identified PMCA 1, 2, 4 in HLE B-3 cells. The apparent MW for PMCA1, 2, 4 was 153.8, 153.5 and 152.9 kDa respectively in 8 cm gel, and 153.1,125.5 and 147.4 kDa respectively in 20 cm gel. PMCA2 probably had some kinds of degradation during the long electrophoresis time in 20 cm gel.
Yang
Hui
H
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
Zeng
Junwen
J
D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004847:Epithelial Cells / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis; D007908:Lens, Crystalline / Q000166:cytology*; D008970:Molecular Weight; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000032:analysis*
D007527:Isoenzymes; D053499:Plasma Membrane Calcium-Transporting ATPases