Ni nta agarose

Fluorescence of LprA-AcGFP1 was observed using an inverted fluorescence microscope (IX-83, Olympus) with a 100× oil immersion objective lens (UPLSAPO100XO, NA 1.4, Olympus) and an sCMOS camera (Prime95B, Photometrics). AcGFP1 was excited by a 130 W mercury light source system (U-HGLGPS, Olympus) with a fluorescence mirror unit U-FGFP (Excitation BP 460–480; Emission BP 495–540, Olympus). Fluorescence image processing was performed with the ImageJ version 1.53 software (National Institutes of Health).
The number of LprA-AcGFP1 at the cell pole was estimated by comparing the fluorescence spot intensity at the pole with the fluorescence intensity of a single His-AcGFP1 molecule as previously reported^{36 (link)}. His-AcGFP1 was purified from E. coli C41(DE3) cells carrying pET19b/ His-AcGFP1 using Ni–NTA agarose (Fujifilm Wako). 10 pg/ml of His-AcGFP1 solution was applied to a coverslip washed by 0.1 M KOH and observed by fluorescence microscopy. In the fluorescent images, a rectangular mask for the fluorescent spot of 30 × 30 pixels was applied to the ROI (region of interest). We defined the spot intensity as the sum of all pixel values within the rectangular mask after subtracting the total background intensity from each pixel value. The number of LprA-AcGFP1 per pole was estimated as the intensity of the cell pole divided by the average intensity of a single His-AcGFP1 molecule.

The pET20b(+)−based human factor IX EGF1 expression plasmid was described [66 (link)]. The pET20b(+)−based human THBS1-TSR3 expression plasmid was described [67 (link)]. Recombinant EGF repeat and TSR proteins were expressed in Escherichia coli BL21(DE3) cells and purified by Ni-NTA agarose (Wako) and RP-HPLC as previously described with a slight modification [68 (link)].

Recombinant GST- or His₆-tagged proteins were expressed in and purified from E. coli. The BL21(DE3)pLysS bacterial cells were transformed with pGEX-6P- or pET30-based vectors, cultured, and then exposed to 0.5 mM isopropyl-β-d-thiogalactopyranoside for 16 h at 10 °C. The cells were then subjected to ultrasonic treatment, and the soluble fraction of cell lysates was purified. The expressed GST-tagged proteins were purified with glutathione-Sepharose 4B beads (Amersham Biosciences) and were eluted from the beads with reduced glutathione. The expressed His₆-tagged proteins were purified with Ni-NTA agarose (Wako) and were eluted with imidazole (Wako). Purified proteins were concentrated with an Amicon Ultra device (Merck Millipore, Tokyo, Japan) and dialyzed against PBS with a Mini Dialysis Kit (GE Healthcare).