Protein Expression Analysis of Germline Stem Cells

The immortalized human male germline stem cells and human primary SSCs were lysed with RIPA buffer (Santa Cruz) for 30 min on ice. Cell lysates were cleared by centrifugation at 12,000 g, and the concentrations of total proteins were measured by BCA kit (Dingguo Company, China). Twenty micrograms of cell lysates from each sample were resolved by SDS-PAGE (Bio-Rad Laboratories, Richmond, CA), and Western blots were performed according to the protocol as described previously1 (link). The chosen antibody included SV40 (Santa Cruz), PCNA (Santa Cruz), RET (Santa Cruz), UCHL1 (AbD Serotec), and ACTB (Proteintech). After extensive washes in PBS, the blots were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad, Hercules, CA).

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Hou J., Niu M., Liu L., Zhu Z., Wang X., Sun M., Yuan Q., Yang S., Zeng W., Liu Y., Li Z, & He Z. (2015). Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation. Scientific Reports, 5, 16922.

Publication 2015

RIPA buffer SDS-PAGE PCNA UCHL1 Chemi-Doc XRS

Antibody Buffer Cell Centrifugation Chemiluminescence Germline cells Human Human male Pcna Proteins Ripa Sds page Stem Sv40 Uchl1 Western blots

Corresponding Organization :

Other organizations : State Key Laboratory of Oncogene and Related Genes, Shanghai Jiao Tong University, Shanghai First People's Hospital

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Variable analysis

independent variables

Immortalized human male germline stem cells
Human primary SSCs

dependent variables

Protein expression levels of SV40, PCNA, RET, UCHL1, and ACTB

control variables

RIPA buffer (Santa Cruz) used for cell lysis
Centrifugation at 12,000 g to clear cell lysates
Protein concentration measurement using BCA kit (Dingguo Company, China)
SDS-PAGE (Bio-Rad Laboratories, Richmond, CA) for resolving cell lysates
Western blotting protocol as described previously1 (link)
Antibodies used: SV40 (Santa Cruz), PCNA (Santa Cruz), RET (Santa Cruz), UCHL1 (AbD Serotec), and ACTB (Proteintech)
Chemiluminescence detection (Chemi-Doc XRS, Bio-Rad, Hercules, CA)

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