deae affigel blue column  (Bio-Rad)

Journal: PLoS ONE

Article Title: Characterization of a Soluble Phosphatidic Acid Phosphatase in Bitter Melon ( Momordica charantia )

doi: 10.1371/journal.pone.0106403

Figure Lengend Snippet: (A) Denaturing gel analysis of the purified PAP fractions from Affigel Blue column by SDS-PAGE and stained by silver nitrate. Lanes 1 and 11, Bio-Rad Precision plus protein standards; lanes 2–4, 0.28, 0.55 and 0.83 µg of fraction #4; lanes 5–7, 0.19, 0.37 and 0.56 µg of fraction #5; lanes 8–10, 0.30, 0.60 and 0.90 µg of the pooled fractions 11–17. (B) PAP activity determined by in-gel phosphatase assay with DiFMUP. Lanes 1 and 8, positive control B-phycoerythrin; lanes 2–7, 0.5, 1, 3, 6, 10 and 12 µg of PAP from the pooled fraction. (C) Native gel analysis of the purified PAP fractions stained with Coomassie Blue. Lanes 1 and 8, native marker unstained protein standards; lanes 2–7, 0.5, 1, 3, 6, 10 and 12 µg of PAP from the pooled fraction.

Article Snippet: Enzyme fraction from Affigel Blue column was concentrated in a Centricon concentrator (Amicon, centrifugal filter devices, Millipore Corporation) and during the course the imidazole buffer was exchanged to protease digestion buffer (50 mM ammonium carbonate, pH 8.0).

Techniques: Purification, SDS Page, Staining, Activity Assay, Phosphatase Assay, Positive Control, Marker