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A Rapid Method for the Purification of Analytical Grade Proteins From Plants Using Preparative SDS-PAGE and Preparative IEF

ample loading, UV-detection, and fraction collection were all done using Bio-Rads Econo System low pressure chromatography workstation. The affinity column was washed with 2 bed volumes of buffer B, followed by 10 ml of 1 mM GTP in buffer B. The column was developed with 10 ml of 3 mM ATP at a flow rate of 1 ml/min. Fractions eluted with 3 mM ATP were pooled and then concentrated using an Amicon Ultrafiltration Cell with a PM30 membrane (30,000 MW cutoff).

Preparative SDS-PAGE
Preparative SDS-PAGE was run according to the discontinuous buffer system of Laemmli (1970).2 A sample was prepared by mixing 3.75 mg of concentrated affinity purified protein with 2x SDS sample buffer at a 1:1 ratio (v/v) and heating at 95 C for 2 minutes. A total sample volume of 2.5 ml (1.5 mg protein/ml) was applied to a 7.5% polyacrylamide separating gel poured to 5.5 cm height and a 1 cm high stacking gel consisting of 4% polyacrylamide. The separating and stacking gels were polymerized in a 37 mm diameter Model 491 Prep Cell casting tube assembly.

Running Conditions
The upper and lower electrophoresis chambers and the elution reservoir of the Model 491 Prep Cell were filled with a 1:10 dilution of Bio-Rads 10x Tris/Glycine/SDS buffer. The lower chamber buffer circulated through the cooling core of the Model 491 Prep Cell at 100 ml/min at room temperature. The Model 491 Prep Cell was run at a constant current of 40 mA for the first 3 hours and at 60 mA constant current for the remainder of the electrophoretic separation. The dye front eluted from the gel at approximately 5 hours of run time. Starting at the dye front, fractions of 2.5 ml were collected at an elution rate of 0.5 ml/mi
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