An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

Abstract

Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagencontaining gels interfered with detection of the p65 protein (one of the subunits in the NF-jB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serumfree culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.

Activating transcription factor 5 enhances radioresistance and malignancy in cancer cells

“…One or two days after seeding, cell lysates were prepared using an improved method as previously reported [39]. To detect proteins from cells cultured in collagen gel-overlay conditions, 5 × 10 4 cells were seeded on a culture dish (radius of 17.5 mm) filled with 500 μL of collagen gel.…”

“…The dish was then filled with culture medium and incubated at 37°C. After 1 day, cell lysates were prepared using an improved method as previously described [39]. Western blotting and quantification of protein expression were performed as previously reported [39, 40], but with blocking for 2 h to detect ATF5.…”

Filamin B Enhances the Invasiveness of Cancer Cells into 3D Collagen Matrices

“…Twenty-four hours after seeding, 450 μL of collagen gel solution was poured onto the cells and incubated in a humidified incubator at 37°C with 5% CO 2 . Then, the cells were cultured for 24 h. Cell extractions were prepared as we previously described (Ishihara et al, 2014). Cells were fixed with 10% trichloroacetic acid in PBS for 3 min on ice and then treated with 0.1% collagenase-L (Nitta Gelatin) in PBS for 90 min at 37°C with 5% CO 2 to digest the collagen gel.…”

Glycosphingolipid GM2 Induces Invasiveness in Irradiation-tolerant Lung Cancer Cells

“…Cells (2×10 5 ) were seeded on a non-coated plastic dish. One to three days after seeding, cell lysates were prepared by an improved method, as previously reported (Ishihara et al, 2016). Briefly, the cells were fixed with 10% trichloroacetic acid in PBS for 3 min on ice and washed three times with PBS on ice.…”