cpCK2 | Casein kinase II subunit alpha (chloroplastic)

Application example

Recombinant cpCK2 proteins wotjpit transit peptide were expressed in DH5a cell line by adding IPTG. Lysates were extracted at 4, 8 and 16hrs induction ​Cells were cultured at RT for 4, 8 and 16hours after IPTG input and were spin-down as indicated time. Cells were washed with cell wash buffer (50mM MOPS pH7.5, 150mM NaCl) and spin-down again. Pellets were resuspended with 5mL of cell wash buffer and sonicated for 10sec, 5times. After centrifugation, supernatants were considered lysates and used for immunoblot assay​ 5uL of lyate were combined with 10uL of SDS loading buffer and denatured at 90’C for 5min. Proteins were loaded and separated on 10% SDS PAGE gel and blotted 1hr to PVDF membrane using tank transfer. Blots were blocked with 2% of gelatin (Sigma) for 1hr at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 for overnight with agitation in PBST. Blots were washed with PBST for 10 min, 3 times at RT with agitation. Blot was incubated in secondary antibody (Alexa Fluor 680 goat anti-rabbit IgG) diluted to 1:10,000 for 1hr at RT with agitation. The blot was washed as above and scanned using Li-COR (Odissey CLX). ​

Courtesy of ​Dr Sang Yeol Kim, University of Illinois, USA