application example

Following SDS-PAGE (12% Seperation Gel) the Blot (PVDF) was stained with PonceauS (A, lowest panel), divided in two halves, scanned and blocked with 5 % not-fat milk powder (Marvel) o.n. at 4 °C. The halves were then incubated separately at RT for 1 h with anti-NpHR or pre-immune serum at the indicated dilutions, rinsed briefly twice with TBS-T (pH 7.4), then washed three times for 15 min, incubated for 1 h at RT with goat anti-rabbit-HRP conjugated secondary antibody (Agrisera, # AS09 602) in a dilution of 1:100 000 and washed as described before. The halves were combined for development with TMA-6 (Lumigen) at the indicated exposure times using Kodak X-ray Film (# 8143059)(A). The anti-NpHR blot was stripped at 70 °C for 30 min and re-probed with anti-Myc (Abcam, # ab9106) and anti-rabbit-HRP (Agrisera, # AS09 602, 1:200000) as described below (B). Samples loaded in the order as indicated above the blot were 9 μg (12 μg in case of HsHR; Amidoblack Assay) of total membrane fractions of yeast expressing: EV = empty vector control; NpHR = Halorhodopsin from Natronomonas pharaonis; HsHR = Halorhodopsin from Halobacterium salinarum; HsBR = Bacteriorhodopsin (D85T) from Halobacterium salinarum;NpHr, HsHR and HsBR include a C-terminal Myc tag. The calculated molecular weights are NpHR-Myc: 33.6 KDa; HsHR-Myc: 31.4 KDa; HsBR-Myc: 29.4 KDa.

Courtesy of Dr. Annegret Honsbein, University of Glasgow, United Kingdom