The assay was performed by mixing stalled take-off complexes (0.1 μM) with EF-G (0.1 μM) and GTP (10 μM) containing trace amounts of [γ-32P]GTP. Reactions were incubated at 37°C, and aliquots were taken at indicated time intervals and quenched with one volume of 50% formic acid. Samples were analyzed by thin-layer chromatography (Polygram CEL 300, Macherey-Nagel) using a 0.5 M potassium phosphate (pH 3.5) running buffer. Radioactivity was detected using a phosphoimager system. The reaction was carried out under initial velocity conditions, as GTP consumption did not exceed 20% at any reaction point. The extent of bypassing was measured in parallel using the [14C]Leu incorporation assay carried out at exactly the same concentrations as the GTPase assay but with addition of EF-Tu–GTP–[14C]Leu-tRNALeu (0.3 μM). The bypassing efficiency (20%) is somewhat lower than in all other experiments due to limiting concentrations of EF-G (0.1 μM instead of 2 μM) and GTP (10 μM instead of 1 mM) used in these experiments, which were chosen to maximize the sensitivity of the assay to GTP hydrolysis.
Polygram cel 300
Manufactured by Macherey-Nagel
Sourced in Ireland
Polygram CEL 300 is a laboratory equipment designed for the separation and analysis of organic compounds using thin-layer chromatography (TLC) technique. It provides a consistent and reproducible platform for performing TLC separations.
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Lab products found in correlation
Zorbax ods column, by Agilent Technologies (1 mentions)
P toluenesulfonic acid, by Merck Group (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Methyl red, by Merck Group (1 mentions)
Pararosaniline base, by Merck Group (1 mentions)
Bromocresol green, by Merck Group (1 mentions)
Thymol blue, by Merck Group (1 mentions)
2 methoxyethanol, by Merck Group (1 mentions)
Propylene glycol methyl ether acetate, by Merck Group (1 mentions)
Bromphenol blue, by Merck Group (1 mentions)
Polyethylene glycol tert octylphenyl ether, by Merck Group (1 mentions)
3 protocols using polygram cel 300
1
GTPase Assay for Ribosomal Translocation
2
Anthocyanin Extraction and Analysis
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The fine powder of the pericarp and aleurone layers of the Millo Corvo kernels (obtained using a manual electric drill) was boiled at 100°C in 2 ml of 2N HCl for 40 minutes. After adding 1 ml of isoamyl alcohol, the upper phase was dried and suspended in EtOH 95% and HCl 1% for the TLC analysis and in methanol for the HPLC run. For TLC analysis, cyanidin, pelargonidin and delphinidin standards were loaded together with the extracts on a pre-coated plastic sheet (Polygram Cel 300, Macherey-Nagel) for TLC using formic acid: HCl: water 5:2:3 as solvent. Developed plates were dried and pictured with a digital camera (A430 Canon) using both white and UV illumination.
For HPLC 20 μl of the sample were injected in an HPLC Kontron Instrument 420 system equipped with a C18 column Zorbax ODS column, 250 mm X 4.6 mm, 5 μm, Teknokroma (Agilent Technologies, Santa Clara, CA, USA) and the absorbance at 530 nm was monitored. Anthocyanins quantification was performed by the method used by Astadi [27 ]; the HPLC conditions were as follows: from min 0 to 8 min, solvent A (10% formic acid) from 96 to 85%, solvent B (100% Acetonitrile) from 4 to 15%; from min 8 to 25, solvent B was kept at 15%; from min 25 to 27, solvent A 20%, solvent B 80%; from min 27 to 30, solvent A 80%, solvent B 20%. The flow rate was 1 ml/min.
For HPLC 20 μl of the sample were injected in an HPLC Kontron Instrument 420 system equipped with a C18 column Zorbax ODS column, 250 mm X 4.6 mm, 5 μm, Teknokroma (Agilent Technologies, Santa Clara, CA, USA) and the absorbance at 530 nm was monitored. Anthocyanins quantification was performed by the method used by Astadi [27 ]; the HPLC conditions were as follows: from min 0 to 8 min, solvent A (10% formic acid) from 96 to 85%, solvent B (100% Acetonitrile) from 4 to 15%; from min 8 to 25, solvent B was kept at 15%; from min 25 to 27, solvent A 20%, solvent B 80%; from min 27 to 30, solvent A 80%, solvent B 20%. The flow rate was 1 ml/min.
3
Sensor Fabrication and Storage Protocol
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Bromphenol blue, bromocresol green, thymol blue, methyl red, nitrazine yellow, methyltriethoxysilane, triethoxy(octyl)silane, 2-methoxyethanol, propylene glycol methyl ether acetate, polyethylene glycol tert-octylphenyl ether, pararosaniline base, N,N-dimethyl-4,4’-azodianiline, p-toluenesulfonic acid and sulfuric acid were purchased from Sigma-Aldrich (Arklow, Co. Wicklow, Ireland). Analytical standards were also purchased from Sigma-Aldrich, Ireland. Substrates (Polygram® CEL 300) were obtained from Machery-Nagel GmbH (Düren, Germany). Water used was high purity Milli-Q water (Millipore >18 MΩcm). A vacuum sealer (iLmyh, model number ZS-11W2) and vacuum sealer bags (Culivac, B16025S) were used for sensor storage and shipment.
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