Silica gel f260 tlc plate

Enzymatic activity of IAInos synthase towards IAInos biosynthesis was determined in a total volume of 8 μL containing 25.2 mM HEPES buffer, pH 7.4, 12 mM UDPG, 6.4 mM IAA, 592 Bq [2’-¹⁴C]IAA (2.035 GBq mmol⁻¹; Hartmann Analytic GmBH), 15 mM myo-inositol, 18 mM D-gluconic acid lactone, 4 mM MgCl₂ and 3 μU of recombinant IAGlc synthase, with 3 μL of the supernatant fluid from tissue homogenates. The reaction was stopped after 30 min incubation at 30 °C by drying 4 μL of aliquots on Silica Gel F₂₆₀ TLC plate (Merck). TLC was performed using ethyl acetate: n-butanone: ethanol: water (5: 3: 1: 1, by vol.) as a solvent. For indole compounds visualization, the plate was stained with van Urk-Salkowski reagent (Ehmann 1977 (link)). Bands corresponding to IAInos were excised and placed in a vial with 2 mL EcoLite ( +) scintillation fluid (MP Biomedicals). Radioactivity level was measured in Wallac 1409 liquid scintillation counter (Wallac Oy).

Enzymatic activity of IAGlc synthase was determined in a total volume of 8 μL containing 74.1 mM HEPES buffer, pH 7.4, 10.9 mM UDPG, 5.8 mM IAA, 592 Bq [2’-¹⁴C]IAA (2.035 GBq mmol⁻¹; Hartmann Analytic GmBH, Braunschweig, Germany), 18 mM D-gluconic acid lactone and 3.6 mM MgCl₂, with 3 μL of the supernatant fluid from tissue homogenates. The reaction was stopped after 30 min incubation at 30 °C by drying 4 μL of aliquots on Silica Gel F₂₆₀ TLC plate (Merck, Darmstadt, Germany). TLC was performed using ethyl acetate: n-butanone: ethanol: water (5: 3: 1: 1, by vol.) as a solvent. For indole compounds visualization, the plate was stained with van Urk-Salkowski reagent (Ehmann 1977 (link)). Bands corresponding to IAGlc were excised and placed in a vial with 2 mL EcoLite ( +) scintillation fluid (MP Biomedicals, Irvine, CA, USA). Radioactivity level was measured in Wallac 1409 liquid scintillation counter (Wallac Oy, Turku, Finland).

The effect of divalent ions on the catalytic activity of recombinant IAGlc synthase was investigated in a total volume of 8 μL containing 25 mM HEPES buffer, pH 7.4, 7.5 mM UDPG, 4 mM IAA and 0.016 μCi [2′-¹⁴C]IAA (55 mCi mmol⁻¹, Hartmann Analytic GmBH, Braunschweig, Germany) with 3 μL of the enzyme preparation. The reaction mixture contained 5 mM MgCl₂, 5 mM CaCl₂, 5 mM MnCl₂, 5 mM EDTA or no ions (control). The reaction was stopped after 15 min incubation at 30 °C by drying 4 μL of aliquots on a Silica Gel F₂₆₀ TLC plate (Merck). The rest of the assay was performed as described above.