Thin-layer chromatography (TLC) plates were used to analyze samples and preparative TLC plates were used to isolate molecules of interest. For samples analysis, 40 μL of the ≤ 3 kDa ultrafiltrate were applied on a 5 cm × 10 cm (length × width) silica gel plate (Sigma-Aldrich). For preparative TLC plates, 1 mL of the ≤ 3 kDa ultrafiltrates were applied on a 20 cm × 20 cm (length × width) silica gel plate (Sigma-Aldrich). Samples were separated with a mobile phase composed of 75 % (v/v) chloroform (BDH Inc., Toronto, Ontario, Canada) and 25 % (v/v) methanol (Fisher Scientific) in a saturated-closed glass chamber. Migration was performed at ambient temperature (21 °C ± 3 °C). After migration, plates were air-dried for 5 min and then observed under a ultra-violet A (UV-A) light (Ultra-Lum Inc., Claremont, CA). Areas which correspond to spots of interest were scraped from the silica gel plate and transferred into a microcentrifuge tube. The extracted silica was weighed and molecules were extracted with acetonitrile at a ratio of 1:3 (w/v) for 2 h at room temperature. Samples were then centrifuged at 18,000 g for 5 min and the supernatant was kept for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
Silica gel plate
Manufactured by Merck Group
Sourced in Germany, United States, Japan
Silica gel plates are thin layers of silica gel used for thin-layer chromatography (TLC). Silica gel is a porous, amorphous form of silicon dioxide that acts as a stationary phase for the separation and analysis of various chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
Sea sand, by Merck Group (3 mentions)
Iodine, by Duksan Pure Chemicals (3 mentions)
Cm 10, by Spectroline (3 mentions)
Avance 500 spectrometer, by Bruker (3 mentions)
Silica gel 60n, by Kanto Chemical (2 mentions)
Silica gel 60, by Merck Group (2 mentions)
Ir affinity 1 spectrometer, by Shimadzu (2 mentions)
Uv 2600 spectrophotometer, by Shimadzu (2 mentions)
Primaide, by Hitachi (2 mentions)
Mcp 500 spectropolarimeter, by Anton Paar (2 mentions)
Silica gel, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
D luciferin, by Merck Group (1 mentions)
N hexane, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Glucose cii test, by Fujifilm (1 mentions)
98 protocols using silica gel plate
1
Thin-Layer Chromatography for Molecule Isolation
2
Lipid Profiling of Arthropod Tissues
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Fat bodies from females at the fifth day after blood meal and hemolymph from days 5 and 10 were collected as described above and were subjected to lipid extraction in chloroform (Bligh and Dyer, 1959 (link)). The fat body neutral lipid and phospholipid composition was analyzed by thin-layer chromatography (TLC) on silica gel plates (Merck KGaA, Darmstadt, Germany) (Horwitz and Perlman, 1987 (link); Kawooya and Law, 1988 (link)). The hemolymph neutral lipid composition was analyzed by high performance thin-layer chromatography (HPTLC) on silica gel plates (Merck) using two consecutive solvent systems (Fan et al., 2004 (link)). The plates were stained with copper reagent as described (Majerowicz et al., 2013 (link)), and the lipid relative composition was determined by densitometry using TotalLab Quant v11 (TotalLab Ltd., Newcastle, United Kingdom) with background corrections, after comparison with commercial lipid standards (Sigma Aldrich).
In order to evaluate hydrocarbon content in the eggshell surface, recently laid eggs were collected, emptied of yolk content with the help of tweezers, and washed in PBS. Groups of 20 eggshells were subjected to two consecutive extractions with 0.5 ml of hexane (Fan et al., 2008 (link)), extracted lipids were analyzed by TLC (Kawooya and Law, 1988 (link)), and the hydrocarbon spot was analyzed by densitometry, as described above.
In order to evaluate hydrocarbon content in the eggshell surface, recently laid eggs were collected, emptied of yolk content with the help of tweezers, and washed in PBS. Groups of 20 eggshells were subjected to two consecutive extractions with 0.5 ml of hexane (Fan et al., 2008 (link)), extracted lipids were analyzed by TLC (Kawooya and Law, 1988 (link)), and the hydrocarbon spot was analyzed by densitometry, as described above.
3
Analysis of Phenolic Compounds in Plant Extracts
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The dried and pulverized plant material (3.0 g) was extracted three times with 80% aqueous methanol (30 mL) under reflux and the resulting extracts were dried under a rotary evaporator. The extracts were subjected to thin-layer chromatography (TLC) analysis for the presence of phenolic compounds. The samples were applied on 20 × 10 cm2 silica gel plate (Merck, Darmstadt, Germany), which was developed in a mobile phase consisting of ethyl acetate-acetic acid-water (8:1:1, v/v/v) and sprayed with 0.1% NA (aminoethyl diphenylborate) ethanolic solution to visualize flavonoids and phenolic acids. The plates were inspected under UV light (366 nm). Additionally, the callus extract was applied on 10 × 10 cm2 cellulose plate (Merck, Darmstadt, Germany) and the two-dimensional chromatogram (2D-TLC) was developed. The first mobile phase consisted of n-butanol-acetic acid-water (4:1:5), while 15% aqueous solution of acetic acid was used as the second mobile phase. After development, the plate was sprayed with 1% ethanolic AlCl3 solution and inspected under UV light (254 nm).
4
PI3K Kinase Inhibition Assay
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The in vitro PI3K kinase assay has been described previously [36 (link)]. Briefly, active PI3K (100 ng) was incubated with taxifolin (0, 20, 40, or 80 mmol/L) or positive control LY294002 (20 mmol/L) for 10 min at 37°C. Phosphatidylinositol (0.5 mg/mL, MP Biomedicals) was added and the mixture was incubated for 5 min at room temperature (RT). This was followed by the addition of reaction buffer (10 mmol/L Tris-HCl (pH7.6), 60 mmol/L MgCl2, and 0.25 mmol/L ATP, 10 mCi γ-32PATP) and incubation for 10 min at 37°C. Termination buffer (1 part 4 N HCl, 3 parts 1 : 1 chloroform: methanol) was added to stop the reaction. After mixing, the lower (chloroform) phase was spotted onto a silica gel plate (Merck KGaA) and 32P-labeled phosphatidylinositol-3-phosphate (PIP3) was resolved by thin-layer chromatography and visualized by autoradiography.
5
TLC Analysis of Bacterial Extracts
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TLC was performed to analyze extracts of bacterial culture broth with organic solvents. Each organic extract was spotted at the bottom of a silica gel plate (20 × 20 cm; Merck) and then placed in a shallow pool of a mixture of chloroform, methanol, and acetic acid (7:2.5:0.5, v/v) as an eluent in a development chamber. The solvent was then allowed to run by capillary action until it reached to the top end of the plate. The silica gel plate was then removed and dried. Separated components were stained with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea). To separate compounds, a preparatory TLC (20 × 20 cm, Merck) was performed using the same eluent. Target spots were scratched and dissolved in methanol.
6
Thin Layer Chromatography of Luciferin
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To confirm the presence of luciferin in the reaction, thin layer chromatography (TLC) was also performed. For this purpose, commercial luciferin (Promega) was used as a standard for comparison with the luciferin obtained from the reaction of p-benzoquinone and cysteine. On a silica gel plate (Merck), 1 μL of 0.1 mM d -luciferin, 1 μL of the mixtures of benzoquinone with l -cysteine or d -cysteine were spotted. The mobile phase was ethyl acetate; ethanol and H2O (5: 2: 3). After chromatography, the fluorescent bands were visualized upon irradiation with UV light and the migration coefficients of the samples were calculated.
7
Qualitative Assay of Phenolic and Flavonoid
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The qualitative assay of phenolic was determined by putting 50 mg sample into test tube and extracted by using vortex for 3 min and 1 ml ethanol as solvent. Ethanol phase was aspirated then 20 ul sample was spotted on silica gel plate (Merck, Darmstadt, Germany) including gallic acid (Sigma Aldrich, St. Louis, USA) as comparation. The plate was put on saturated chamber (CAMAG, Muttenz, Switzerland) with mobile phase, the mix solution of 9.5 ml methanol (Merck, Darmstadt, Germany) and 0.5 ml formic acid 10% (Merck, Darmstadt, Germany), then elute until the border. The plate was dry off, observed under UV light (Sankyo Denki, Kanagawa, Japan), and spray using ferri chloride reagent (Merck, Darmstadt, Germany). Subsequently, the same methode was run for the flavonoid, with the modification of the comparation (quercetin) and the mobile phase, the mix solution of 6.0 ml n-hexane (Merck, Darmstadt, Germany), 4.0 ml ethyl acetate (Merck, Darmstadt, Germany), and 0.1 ml formic acid (Merck, Darmstadt, Germany).
8
Chitinase Kinetics and Hydrolysis Products
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The kinetic parameters of chitinases in colloidal chitin was determined by measuring enzymatic activity at 50 °C in optimum pH for 30 min with substrate concentrations of 0.05%, 0.1%, 0.2%–0.8% (at interval of 0.2%), 1.0%, and 2.0%. The Km and Vmax values were calculated from kinetic data fitting of the Michaelis–Menten equation [69 (link)].
The hydrolysis products of chitinases were determined by thin layer chromatographic (TLC) method using colloidal chitin and (GlcNAc)2-4 as substrates [70 (link)]. Briefly, purified chitinase and 1% (w/v) colloidal chitin or (GlcNAc)2-4 were mixed in optimum pH, and then the mixtures were incubated at optimum temperature (50 °C) for 10 min, 30 min, 1 h, 2 h, or 4 h. The reaction products were spotted onto a Silica gel plate (Merck, Damsladt, Germany), and spread using butanol: water: ammonia water (6:3:1, v/v/v) as spreading agent. The plate was sprinkled with chromogenic agent (containing 4 g diphenylamine, 4 mL aniline, 2 mL concentrated hydrochloric acid, 20 mL phosphate, and 200 mL acetone).
The hydrolysis products of chitinases were determined by thin layer chromatographic (TLC) method using colloidal chitin and (GlcNAc)2-4 as substrates [70 (link)]. Briefly, purified chitinase and 1% (w/v) colloidal chitin or (GlcNAc)2-4 were mixed in optimum pH, and then the mixtures were incubated at optimum temperature (50 °C) for 10 min, 30 min, 1 h, 2 h, or 4 h. The reaction products were spotted onto a Silica gel plate (Merck, Damsladt, Germany), and spread using butanol: water: ammonia water (6:3:1, v/v/v) as spreading agent. The plate was sprinkled with chromogenic agent (containing 4 g diphenylamine, 4 mL aniline, 2 mL concentrated hydrochloric acid, 20 mL phosphate, and 200 mL acetone).
9
Saccharide Profiling by HPTLC
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Samples XY01-XY14 and XY15-XY22 were dissolved in H2O at concentrations of 10 mg/mL and 2 mg/mL, respectively. Dextran (5 mg/mL) and xylan (5 mg/mL) were hydrolyzed by using 1 M TFA at 80 °C for 4 h to produce the oligosaccharides ladders. Saccharide profiling based on HPTLC was performed according to our previously reported method [28 (link),29 ]. In brief, all the samples (4 μL) were applied on a 20 cm × 10 cm silica gel plate (Merck, Darmstadt, Germany) by using an AS30 HPTLC Applicator (Desaga GmbH, Germany). The bands were 8 mm wide, 13 mm distance, and 10 mm from the bottom edge. Then, the plates were developed for two times with 1-butanol/isopropanol/acedic acid/water (7:5:1:2, V/V/V/V) as mobile phase. Finally, the developed plates were sprayed with aniline-diphenylamine-phosphoric acid solution and heated at 105 °C on a YOKO-XR plate heater (Wuhan YOKO Technology Ltd., China) for 15 min; then the plates were covered with glass and photographed. After that, the plates were scanned at λ = 520 nm using densitometer CD60 (Desaga GmbH, Germany).
10
Isomaltase Activity Assay and Hydrolysis Analysis
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Isomaltase activity was assayed at 30 °C using 0.1 % isomaltose as the substrate in 40 μL 100 mM sodium phosphate buffer (pH 6.0). After stopping the reaction by brief heating, the amount of glucose was determined by the glucose oxidase method using the Glucose CII-Test (Wako Pure Chemicals Industries Ltd., Osaka, Japan).13) (link) Hydrolysis of pNPαG was examined in a manner similar to that described above, except that the reaction was stopped by adding 200 μL sodium carbonate and the color shift was determined at 405 nm. For TLC, 1 μL reaction mixture was spotted onto a silica gel plate (Merck KGaA, Darmstadt, Germany), which was then developed using a solvent system comprising water:1-butanol:2-propanol (4:3:12). The sugars were visualized using 2 M sulfuric acid containing 0.2 % orcinol.
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