Journal: Cell Transplantation

Article Title: Human Umbilical Cord Blood Serum–derived α-Secretase

doi: 10.1177/0963689718759473

Figure Lengend Snippet: Fractionation of amyloid precursor protein (APP)-specific α-secretase activity in cord blood serum (CBS). To purify and eventually identify the α-secretase activity in CBS, the Econo-Pac Serum IgG Purification Kit (Bio-Rad, Philadelphia, PA, USA) was initially employed to remove highly abundant IgG. CBS was then desalted using Econo-Pac 10DG columns. The desalted serum was applied to DEAE Affi-Gel Blue columns to remove residual IgG and collect 20 additional protein fractions, by eluting with an increasing ionic strength gradient of NaCl buffer ranging from 0.1 M to 2.0 M. The remaining proteins on the column were eluted by the regeneration buffer included in the kit and collected as the regeneration fraction (Reg). (A) Chinese hamster ovary cells stably expressing wild-type human APP (CHO/APPwt) cells were cultured in 24-well plates and treated with 10 μL of each protein fraction for 2 h. Conditioned media were then collected and analyzed by soluble amyloid precursor protein α (sAPPα) Western blot (upper panel) and ELISA (lower panel). 10 μL CBS, desalted CBS, and phosphate-buffered saline (PBS; Ctrl) were included under the same cell culture conditions as positive and negative controls, respectively. Cell lysates were also prepared from each fraction-treated cell culture as an additional reference to evaluate sAPPα production levels (data not shown). (B) Protein concentration of each fraction. (C) CHO/APPwt cells were treated with the 0.6 to 1.0 M NaCl-eluted fractions from 10 different CBS lots, as well as whole CBS and PBS (Ctrl), for 2 h and the conditioned media were collected for sAPPα ELISA. The results were presented as mean (± SD ) sAPPα produced (ng/mg protein). In addition, each protein fraction was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess total protein fractionation (C, right panel).

Article Snippet: Initially, removal of highly abundant immunoglobulins and desalting were performed using Bio-Rad Econo-Pac Serum IgG Purification Kit and 10DG columns.

Techniques: Fractionation, Activity Assay, Purification, Stable Transfection, Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Saline, Protein Concentration, Produced, Polyacrylamide Gel Electrophoresis