ion exchange membrane column  (A&A Biotechnology)

Journal: The Journal of Cell Biology

Article Title: Identification of prothymosin-α1, the necrosis–apoptosis switch molecule in cortical neuronal cultures

doi: 10.1083/jcb.200608022

Figure Lengend Snippet: Purification and identification of ProTα. (a) Scanning EM (SEM) analysis of neuronal necrosis. Cortical neurons were cultured at LD (10 5 cell/cm 2 ) under the serum-free condition. (b) Parallel time-dependent decreases in survival activity and increases in PI staining (10 μg/ml) after the start of LD and serum-free cultures. Survival activity was evaluated by the WST-8 reduction activity. (c) Parallel CM concentration-dependent increases in survival activity and decreases in PI staining. The activities were measured at 12 h after the start of LD and serum-free cultures with various concentrations of CM. Error bars indicate mean ± SEM. (d and e) Purification of ProTα. Vivaspin 2, and Vivapure Q mini were used for ultrafiltration (d) and ion-exchange spin column chromatography (e), respectively. The samples indicated by asterisks in panels d and e were used for further separation in panels e and f, respectively. (f) SDS-PAGE analysis of the final purified material. (g) Predicted amino acid sequence.

Article Snippet: The CM was first subjected to ultrafiltration (Vivaspin 2; Sartorius KK), and the active materials observed in the >5-kD fraction were applied to an ion-exchange membrane spin column (Vivapure Q Mini; Sartorius KK), which had been equilibrated with 20 mM sodium acetate, pH 5.2.

Techniques: Purification, Cell Culture, Activity Assay, Staining, Concentration Assay, Column Chromatography, SDS Page, Sequencing