Anti-V5 | V5 epitope tag

Samples:
1 – 10 μg protein from non induced E. coli cells transformed with the expression plasmid
2 – 10 μg protein from IPTG-induced E. coli cells extracted with protease inhibitors (EDTA and PMSF)
3 – 10 μg protein from IPTG-induced E. coli cells extracted without protease inhibitors
4 – MW markers (Sigma C1992)

10 of total protein extracted from IPTG-induced or non induced Escherichia coli cells harvested and stored at -20°C, in 50 mM Tris-HCl buffer, pH 7.5; following centrifugation at 1 500 g, extracts were denatured with the same volume of 2X treatment buffer consisting of 125 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol and 10% (v/v) β-mercaptoethanol at 95°C for 10 min. Samples were separated at RT on a discontinuous SDS-PAGE (10% separating gel, 5% stacking gel) and blotted overnight to nitrocellulose (pore size of 0.45 μm), using wet transfer at RT. Blot was blocked with 3% BSA for 1 h at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 for 90 min at RT with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed twice for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG alkaline phosphatase conjugated) diluted to 1: 7 500 in TBS-T for 90 min at RT with agitation. The blot was washed three times as above and equilibrated for 5 min in AP-buffer (0.1 M Tris-HCl, pH 9.5, containing 0.1 M NaCl and 5 mM MgCl2). Bands were developed in a freshly-prepared reaction mixture consisting of 0.3 mg mL-1 nitro blue tetrazolium (NBT) and 0.4 mg mL-1 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in AP-buffer. The reaction was allowed to proceed for 7.5 min, and then blocked with 20 mM EDTA in TBS-T.

Courtesy of Dr. Giuseppe Forlani, University of Ferrara, Italy