Silica gel h thin layer plate

Expression plasmids containing nothing (pPJ131), alfi_1465 (pAf1465), or SulA (pSulA) were transformed into E. coli strain Top10 cells (Invitrogen), and these strains were grown in 100 ml terrific broth medium at 37 °C and 200 rpm. Cells were harvested by centrifugation (10,000g for 10 min) at A₆₀₀ = 4.0 and the cell pellets were washed in PBS, then H₂O, and then resuspended in 1 ml of 150 mM NaCl, 100 mM Tris, pH 7.2. Cells were broken by two passes through a French press at a working pressure of 20,000 psi, and cell free bacterial lysates were separated from cellular debris by centrifugation (20,000g for 30 min). The protein concentration of each lysate was determined by Coomassie Protein Assay (Thermo Scientific). SulA activity assays were carried out at 30 °C for 30 min in 20 μl reactions containing 500 ng lysate, 200 μM [¹⁴C]16:0-ACP, 1% Triton X-100, 100 mM potassium phosphate buffer, 150 mM NaCl, pH 7.6, with and without 20 mM cysteate. The entire reaction was spotted on a Silica Gel H thin-layer plate (Spectrum Chemical) and separated using ethanol:chloroform:triethylamine:H₂O (34:30:35:6.5, v/v). Product formation was visualized using a Typhoon PhosphorImager and quantified using ImageQuant (Cytiva Life Sciences).

The OhyA, OhyA(E82A), and OhyA(Y201F) activities were assayed as described previously (15 (link)). Briefly, 20 μl reactions containing 50 mM potassium phosphate buffer, pH 6.0, 150 mM NaCl, 10 mM DTT, 50 μM FAD (Millipore Sigma, Cat# F6625), 0.2 mg/ml fatty acid-free bovine serum albumin (Millipore Sigma, Cat# A6003), 20 μM [1-¹⁴C]oleate (specific activity 54.3 mCi/mmol) (PerkinElmer, Cat# NEC317250UC), and 1 μg OhyA, 100 μg OhyA(E82A), or 10 μg OhyA(Y201F) were incubated at 37 °C for 20 min. These enzyme concentrations were experimentally determined based on prior reactions with varying enzyme concentration monitoring detectable hydroxy fatty acid product formation. A 10 μl aliquot of the reactions were spotted on a Silica Gel H thin-layer plate (Spectrum Chemical, Cat# 476–10,450) and separated using chloroform:methanol (90:10, v/v). Hydroxy fatty acid product formation was visualized using a Typhoon PhosphorImager and quantified using ImageQuant (Cytiva Life Sciences, https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software).