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300 Lee and Aguilar
Abs 280
PLA 2 Activity
(CPM)
12000
0.1 AU
8000
4000
0.00
0 10 20 30 40 50 60 70 80
(min)
Fig. 2. Elution of proteins in the Sigma PLA 2 using sodium octanesulfonate and
acetonitrile gradients. Two hundred micrograms of protein in approximately 200 μl is
injected to the phosphatidylcholine immobilized column (4.6 i.d. × 100 mm). Mobile
phase A contained 0.1 M Tris (pH 7.2), 0.2 M KCl, 20% ethyleneglycol, and 0.05%
NaN 3 . Mobile phase B contained 1% sodium octanesulfonate in mobile phase A. Mobile
phase C contained 4% acetonitrile in mobile phase B. The dotted line represents the
chromatography gradient. (•) PLA 2 activity. Each square represents almlchromatographic
fraction assayed for PLA 2 activity. Reproduced with permission from ref. 13.
7. 1 mL fractions are collected from the column. The protein content in each fraction
is determined using the bicinchoninic acid (BCA) protein assay kit (Pierce), and
the purity is further analyzed using SDS–PAGE. A 12% polyacrylamide gel is
routinely used for analyzing the protein species in each collected fractions. Silver
stain is then used to visualize the protein bands.
4. Notes
1. The immobilized phospholipids are labile under acid and base conditions. The
addition of organic acid modifiers such as trifluoroacetic acid and acetic acid into
the separation buffer has to be avoided.
2. The addition of low levels of detergents or lysophospholipids with a high critical
micellar concentration (cmc) of detergent additives is often required to maintain
the activity of the protein of interest. An additive with a low cmc is preferable to
facilitate their subsequent removal by, for example, dialysis.
3. Some proteins and non-protein materials can be strongly retained on the column
and failure to flush out these materials may affect the separation result. It is