Description
Transient overexpression lysate of chloride channel 1, skeletal muscle (CLCN1) with a C-Myc/DDK tag was expressed HEK293T. Components: 1 vial of 100 µg gene specific transient over-expression cell lysate in RIPA buffer 1 vial of 100 µg empty vector transfected control cell lysate in RIPA buffer 1 vial of 250 µL 2×SDS Sample Buffer (4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromphenol blue, 100mM DTT) Preparation: HEK293T cells in 10-cm dishes were transiently transfected with MegaTran Transfection Reagent and 5 µg TrueORF cDNA plasmid. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix (Sigma), 1mM PMSF and 1mM Na3VO4), and then centrifuged to clarify the lysate. Protein concentration was measured by BCA kit. Cell lysates were aliquoted and stored at -20 centigrade before shipping