Measurements of the activity of the recombinant DeDFR1 and DeDFR2 were performed according to the methods of Petit et al. and Katsu et al. with minor modifications [25 (link), 35 (link)]. A 500-μL reaction mixture consisted of 1.6 mM NADPH, 50 mM Tris-HCl (pH 7.0), 0.8 mM substrate, and 5 mg of total protein. Three dihydroflavonols were selected as experimental substrates: dihydrokaempferol (DHK), dihydroquercetin (DHQ) and dihydromyricetin (DHM) (Sigma-Aldrich). Each reaction was terminated after 30 min at 30°C by extraction with ethyl acetate. Because the obtained leucoanthocyanidins were unstable in solution, the residues were dissolved after evaporation in 200 μL of n-BuOH-HCl (95:5) and incubated at 95°C for 30 min to form anthocyanidins. The HPLC analysis of the DFR products was similar to the anthocyanidin detection described above. The process was repeated at least three times for each sample.
Dihydrokaempferol
Manufactured by Merck Group
Sourced in United States
Dihydrokaempferol is a chemical compound used as a reference standard in analytical and biomedical research. It is a flavonoid with the molecular formula C₁₅H₁₂O₅.
Automatically generated - may contain errors
Lab products found in correlation
Dihydroquercetin, by Merck Group (5 mentions)
Naringenin, by Merck Group (4 mentions)
Quercetin, by Merck Group (3 mentions)
Dihydromyricetin, by Merck Group (2 mentions)
Luteolin, by Merck Group (2 mentions)
Epicatechin, by Merck Group (2 mentions)
Dihydromyricetin dhm, by Merck Group (1 mentions)
Cyanidin 3 o glucoside, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Catechin, by Merck Group (1 mentions)
Apigenin, by Extrasynthese (1 mentions)
Isoliquiritigenin, by Merck Group (1 mentions)
Quercetin, by Cayman Chemical (1 mentions)
Kaempferol, by Cayman Chemical (1 mentions)
7 4 dihydroxyflavone, by Extrasynthese (1 mentions)
Liquiritigenin, by Bio-Techne (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Quercetin, by Extrasynthese (1 mentions)
Myricetin, by Extrasynthese (1 mentions)
Kaempferol, by Extrasynthese (1 mentions)
8 protocols using dihydrokaempferol
1
Recombinant DeDFR1 and DeDFR2 Activity Assay
2
Quantification of Flavonoid Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dihydroquercetin (DHQ), dihydromyricetin (DHM) and dihydrokaempferol (DHK) were bought from Sigma-Aldrich (St. Louis, MO, USA) and prepared as 10 mg/mL solutions in methanol (chromatographic grade). Cyanidin 3-O-glucoside for drawing standard curve was also purchased from Sigma-Aldrich and diluted as one mg/mL solutions in methanol (St. Louis, MO, USA).
3
Quantitative Analysis of Flower Flavonoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anthocyanins were determined using high-performance liquid chromatography (HPLC) as previously described (Qi et al., 2013 (link)). For extraction of other flavonoids, freeze-dried flowers were finely ground and 50mg was extracted in 500 μl of MeOH for 48h at 4 °C in darkness. After samples were centrifuged, the supernatants were transferred to fresh tubes and the pellet was resuspended and incubated in 500 μl of 1% MeOH at 4 °C for 24h, and then the supernatant was combined for further HPLC analysis. HPLC was performed as previously described (Qi et al., 2013 (link)). Cyanidin, Cyanidin-galactoside, dihydroquercetin, dihydrokaempferol, (+)-catechin, (–)-epicatechin, luteolin, naringenin, and quercetin were obtained from Sigma-Aldrich China (Shanghai). Standards of afzelechin, (–)-epiafzelechin, (+)-gallocatechin, and (–)-epigallocatechin were purchased from BioBioPha (Yunnan, China). The delphinidin chloride (ChromaDex, Santa Ana, CA, USA), petunidin chloride (ChromaDex), and other flavonoids such as dihydromyricetin (YiFang S&T, Tianjin, China) equivalents were used as standards for quantification. Mean values and SDs were obtained from three biological replicates.
4
Quantitative Analysis of Flavonoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All solvents used for solid‐phase extraction, HPLC analysis/purification and MS‐based experiments were LC‐MS grade from either Sigma‐Aldrich or VWR Chemicals. Authentic standards for LC‐HRESIMS quantification and molecule identification through LC‐HRESIMS and LC‐UV/vis were purchased from different suppliers: p‐coumaric acid (Sigma Aldrich, USA), naringenin (Sigma Aldrich, USA), dihydrokaempferol (Sigma Aldrich, USA), kaempferol (Cayman Chemical, USA), apigenin (Extrasynthese, Genay, France), 2‐hydroxynaringenin (Ambinter, Orléans, France), isoliquiritigenin (Sigma Aldrich, USA), liquiritigenin (Tocris Bioscience, Bristol, UK), (2R,3R)‐garbanzol (BioBioPha, Kunming, Yunnan, China), (2S,3S)‐garbanzol (AnalytiCon Discovery, Potsdam, Germany), resokaempferol (Extrasynthese), fustin (Biosynth Carbosynth, Compton, UK), butin (ChemFaces, Wuhan, China), 7,4′‐dihydroxyflavone (Extrasynthese, France), quercetin (Cayman Chemical) and dihydroquercetin (Sigma Aldrich).
5
Petunia Flower Developmental Stages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flowers (stage 1: buds of 0.6–3 cm length, stage 2: buds of 3–5 cm length, stage 3: open flowers) of cv. Salmon Ray (Danziger, Moshav Mishmar Hashiva, Israel), cv. Viva Orange (Florensis, Ambacht, The Netherlands), and cv. Electric Orange (Selecta One, Stuttgart, Germany) were harvested in the summers 2015–2017. Non-transgenic control plants of Petunia × hybrida cv. BabyDoll were obtained from Selecta One, cvs. Corso Rot, Corso Blau and Blackberry were purchased from Austrosaat (Vienna, Austria). The plant material was harvested from balcony pots or garden beddings, shock-frozen and kept at −80°C until analysis. Images of the petunia varieties are found in Figures 1 , 2 and Supplementary Figure S3 .
Reference compounds (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, dihydromyricetin, dihydroquercetin, kaempferol, myricetin, and quercetin) were purchased from Extrasynthese (Genay, France), dihydrokaempferol from Sigma Aldrich (Vienna, Austria). Radiolabeled substrates were synthesized as previously described (Halbwirth et al., 2006 (link)).
Reference compounds (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, dihydromyricetin, dihydroquercetin, kaempferol, myricetin, and quercetin) were purchased from Extrasynthese (Genay, France), dihydrokaempferol from Sigma Aldrich (Vienna, Austria). Radiolabeled substrates were synthesized as previously described (Halbwirth et al., 2006 (link)).
6
Spectrophotometric Analysis of Plant Metabolites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemical standards (apigenin, luteolin, naringenin, eriodictyol, dihydrokaempferol, dihydroquercetin, kaempferol, quercetin, epicatechin) and HPLC grade solvents were purchased from Sigma (St. Louis, MO). Cyanidin (chloride), pelargonidin (chloride), peonidin (chloride) were purchased from Cayman Chemical (Ann Arbor, MI). Pictures of Arabidopsis seeds and seedlings were taken using a dissecting microscope with a Nikon SMZ1500 camera with a Nikon WD54 HR Plan Apo lens. A BioMate 3S UV-visible spectrophotometer (ThermoFisher Scientific, Waltham, MA) was used to measure absorbance.
7
Tea Cultivar Metabolite Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tea plants (C. sinensis cv. Shuchazao) used in this experiment were grown in an experimental tea field at Anhui Agricultural University, Hefei, China (East longitude 117.27, North latitude 31.86). Leaves at different developmental stages (buds and 1st, 2nd, 3rd and 4th leaves), mature leaves, tender stems and roots were harvested and immediately frozen in liquid nitrogen for further study. The wild-type Arabidopsis thaliana used in our laboratory was ecotype Columbia 0 (Col-0), which was grown in a chamber at a constant temperature of 16 ± 3 °C and a 16/8 h (light/dark) photoperiod. The tobacco (Nicotiana tabacum cv. G28) used for the transgenic assays was provided by the University of Science and Technology of China (Hefei, Anhui, China) and grown in a growth chamber at a constant temperature of 24 ± 3 °C and a 12/12 h (light/dark) photoperiod.
Escherichia coli DH5α and BL21 (DE3) (TransGen Biotech, Beijing, China) were used as the host strain and expression strain for the prokaryotic expression, respectively. Agrobacterium tumefaciens C58C1 and EHA105 were kindly provided by the University of Science and Technology of China.
The substrates DHQ, dihydromyricetin (DHM) and dihydrokaempferol (DHK) and standard C, GC, EC, EGC, ECG, EGCG, procyanidin B2 and cyanidin-3-O-glucosides were purchased from Sigma (St. Louis, MO, USA).
Escherichia coli DH5α and BL21 (DE3) (TransGen Biotech, Beijing, China) were used as the host strain and expression strain for the prokaryotic expression, respectively. Agrobacterium tumefaciens C58C1 and EHA105 were kindly provided by the University of Science and Technology of China.
The substrates DHQ, dihydromyricetin (DHM) and dihydrokaempferol (DHK) and standard C, GC, EC, EGC, ECG, EGCG, procyanidin B2 and cyanidin-3-O-glucosides were purchased from Sigma (St. Louis, MO, USA).
8
Flavonoid Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The enzyme assay was performed using 20 mL E. coli cultures expressing the respective constructs right after induction with L-arabinose. 100 µL substrate (10 mg/mL Naringenin, eriodictyol or dihydroquercetin), 50 µL 2-oxoglutaric acid, 50 µL FeSo4 and 50 µL 1 M sodium ascorbate were added. The cultures were incubated at 28 °C overnight. To extract flavonoids, 1 mL was removed from each culture and mixed with 200 µL ethyl acetate by vortexing for 30 s. After centrifugation for 2 min. at 14,000 g, the organic phase was transferred into a fresh reaction tube. Samples were taken after 0 h, 1 h, 2 h, 3 h, 4 h and 24 h. Flavonoid content was analysed by high-performance thin-layer chromatography (HPTLC). Naringenin (Sigma), dihydrokaempferol (Sigma), kaempferol (Roth), eriodictyol (TransMIT PlantMetaChem), apigenin (TransMIT PlantMetaChem), dihydroquercetin (Roth) and quercetin (Sigma) were dissolved in methanol and used as standards. 3 µL of each methanolic extract were spotted on a HPTLC Silica Gel 60 plate (Merck). The mobile phase was composed of 50 % chloroform, 45 % acetic acid and 5 % water. Flavonoid compounds were detected as described previously (Stracke et al., 2007) (link), using diphenylboric acid 2-aminoethyl ester (DPBA) and UV-light (Sheahan and Rechnitz, 1992) .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!