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All the above procedures prior to boiling in SDS-sample loading buffer were carried out either on ice or in 4°C cold room.
Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer's protocol.
The fatty acid decarboxylation assays containing 0.2 to 1.0 μM OleTJE or OleTJE-RhFRED, 200 μM fatty acid substrate (from C8 to C20), 500 μM H2O2 (for OleTJE) or 500 μM NADPH (for OleTJE-RhFRED) in 200 μl of storage buffer were carried out at 28°C for 2 h.
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A wash step in calcium buffer was carried out before acquisition on a FacsCalibur flow cytometer (Becton Dickinson, Oxford, England).
A wash step in calcium buffer was carried out prior to acquisition on a FACSCalibur flow cytometer (Becton Dickinson).
A wash step in calcium buffer was carried out prior to acquisition on a FacsCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Quantitation of KA in the phosphate buffer was carried out by UV spectrophotometry at a wavelength of 268 nm (Hitachi), using the calibration curve determined in phosphate buffer, demonstrated in Section 3.3.2 to determine the concentration of KA permeate.
Protein extraction in Bradley buffer was carried out from leaves, and skin and flesh of fruit at two stages, green and red, from each transgenic line in the field.
Preparation of buffers was carried out at the appropriate temperatures by mixing the appropriate dibasic and basic salts.
After pre-hybridization overnight at room temperature followed by a 2-hour incubation at 42 °C, hybridization with 200 ng probe/ml buffer was carried out.
The protocol of washing repeat count with Selection buffer, Washing buffer-1 and Washing buffer-2 was carried out according to the selection round program (F1 to F9) as follows: 3 3 3 in F1, 4 5 3 in F2, 5 5 5 in F3, 5 7 5 in F4, 5 7 7 in F5, 5 10 7 in F6, 5 10 10 in F7, 5 15:10 in F8, and 5 15:15 in F9.
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