Linear gradient fractionation procedure

The sample is loaded at a flow-rate of 1 ml/min onto the FPLC column equilibrated with the same MOPS buffer used to resuspend the RNA pellets. The free nucleotides are completely removed with a 5-ml wash with 350 mM NaCl and the RNA is eluted with a 20-ml (350—750 mM NaCl) linear gradient and analyzed by PAGE/urea gel electrophoresis (see later). Up to 2 mg of RNA can be loaded onto and eluted from a 1-ml (of resin) mono Q column without loss of resolution. The homogeneity of RNA in the fractions collected, as seen by gel electrophoresis, should be >90%. The appropriate fractions are pooled and the RNA collected by ethanol precipitation. The RNA pellet is washed twice with 70% ethanol, air-dried, and finally redissolved in DEPC-treated H20. The total recovery after the entire procedure of purification is = 90%. This protocol yields = 800 pmoles of purified 002 mRNA/pmole template DNA. 

PLA2 was modified with PG according to the procedure described by Takahashi (1968). One micromole of PLA2 in 1 ml of 0.1 M sodium borate buffer (pH 9.0) was incubated with 100-fold molar excess of PG. The reaction was allowed to proceed for 2 hr at 37°C, then quenched by the addition of a few drops of acetic acid. The modified proteins were immediately desalted by passing through a Sephadex G-25 column equilibrated with 0.1 M acetic acid and the protein fraction was lyophilized. The modified proteins were separated by HPLC on a SynChropak RP-18 column (4.6 mm x 25 cm), equilibrated with 0.1% TFA and eluted with a linear gradient of 28-35% acetonitrile for 35 min. Flow rate was 0.8 ml/min and the effluent was monitored at 280 nm. 

Cell fractionation Details of the fractionation procedure for cytoplasmic material freed of nuclei and mitochondria has been described in detail in a previous publication [7]. However, a brief description is given in a chart (Figure 1). Figure 2 shows the densities of the sucrose in the linear gradient of each collected fraction. The fractions were collected by a peristaltic pump (Pharmacia Inc.) from the bottom of the tube containing subcellular particles. 

The PEG precipitate (30- 0%) obtained from the 100,000 g supernatant of the epicarp of immature fruits of calamondin was submitted to chromatography on a column of DEAE-Sephacel and developed with a linear gradient of NaCl (0.1-0.2M). As shown in fig. 1, the limonene and selinene cyclase activities were resolved. The electrophoresis of the fractions involved in mono- and sesquiterpene biosynthesis showed the purification obtained after the DEAE-Sephacel column. Incubations of small gel pieces with [ H]-GPP and [ H]-FPP led to the localization of the cyclases involved in both limonene and selinene synthesis and indicated that the two cyclases are well separated on the gel. In order to improve the purification of these cyclases, the active fractions eluted from the DEAE-Sephacel column were submitted to affinity chromatography using Sepharose APP. This procedure contributed to a 90-fold and 70-fold purification for limonene and selinene cyclases respectively. An isoelectric point of 5A 0.2 was found for limonene-cyclase whereas 3-selinene-cy-clase presented a value of 6 0.2. After gel permeation with Sephadex G 200, the same molecular weight of 67 3 kD was attributed to the two cyclases. In addition, prenyltransferases obtained from a DEAE-Sephacel column developed with a step gradient (0.1, 0.2, 0.3 M NaCl) were also isolated on the same affinity column. These prenyltransferases were 160 fold purified. Their molecular weight was about 70 kD and they exhibited an isoelectric point of 3.2. 

The final purification step is a linear biphasic gradient of acidified acetonitrile at a flow rate between 0.08 and 0.25 mL/min (0.08 mL for the microbore column, 0.1-0.25 mL for the narrowbore column). The following two-step gradient for step 2 is used. However, to increase resolution, a column with a smaller internal diameter is needed and column temperature is increased to 37°C. For fraction handling, use the procedure according to Subheading 3.3.2.I., part 4. 

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