Proteins were analyzed using a liquid chromatography instrument (CBM-20A; Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler (SIL-20A), ultraviolet-visible (UV-VIS) (SPD-20A), and a fluorescence detector (RF-20Axs). Molecular weight was determined using SEC with a Malvern Zetasizer µV instrument (Malvern Instruments Ltd.) measuring inline SLS and DLS. Data were processed using Lab Solution Version 5.51 (Shimadzu Corporation) and OmniSec 4.7 (Malvern Instruments Ltd.) software. Samples (50 µg) were injected into the column (Superdex 200 Increase 5/150; GE Healthcare, Uppsala, Sweden) using the autosampler. The column was equilibrated with 50 mM Na3PO4, 150 mM NaCl, pH 7.2 running buffer. The measurements were run with a flow rate of 0.1 mL/min at 20 °C. The monomeric peak of bovine serum albumin (BSA) was used for calibrating the system to calculate molecular weight from the measured SLS intensity.
Labsolution version 5
Manufactured by Shimadzu
Sourced in Japan
LabSolution version 5.53 is a software application developed by Shimadzu for data analysis and instrument control. The software provides a user-friendly interface for managing and processing data generated by various Shimadzu laboratory instruments. It supports a wide range of analytical techniques and is designed to streamline laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Cbm 20a, by Shimadzu (4 mentions)
Frc 10a, by Shimadzu (4 mentions)
Prominence lc 20a system, by Shimadzu (3 mentions)
Omnisec 4, by Malvern Panalytical (2 mentions)
Superdex 200 increase 5 150, by GE Healthcare (1 mentions)
β amylase, by Merck Group (1 mentions)
Superdex 200 5 150 column, by GE Healthcare (1 mentions)
Lc 20ap, by Shimadzu (1 mentions)
Dgu 20a3r, by Shimadzu (1 mentions)
Lc 20adxr pump, by Shimadzu (1 mentions)
Xbridge beh c18, by Waters Corporation (1 mentions)
Spd m20a uv, by Shimadzu (1 mentions)
Prominence lc 20ad system, by Shimadzu (1 mentions)
6 protocols using labsolution version 5
1
Protein Characterization by Liquid Chromatography
2
Molecular Weight Determination of Zebrafish CA VI-PTX
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Molecular weight determination of zebrafish CA VI–PTX was performed using a Malvern Zetasizer μV instrument (Malvern Instruments Ltd., Worcestershire, UK) running static light scattering (SLS) and dynamic light scattering (DLS) methods. Analysis was performed using a liquid chromatography instrument (CBM-20A, Shimadzu Corporation, Kyoto, Japan) equipped with autosampler (SIL-20A), UV–VIS (SPD-20A) and fluorescence detector (RF-20Axs). Data were processed using Lab Solution Version 5.51 (Shimadzu Corporation) and OmniSec 4.7 (Malvern Instruments Ltd., Worcestershire, UK) softwares. A sample of the protein (50 μg) was injected on a Superdex 200 5/150 column (GE Healthcare, Uppsala, Sweden) equilibrated with 50 mM NaH3PO4, 500 mM NaCl pH 8 buffer. Runs were performed with flow rate of 0.1 ml/min at 20 °C using a thermostated cabin. The MW of the zebrafish CA VI–PTX was determined either by using a standard curve based on MW standard proteins (SEC analysis; CA 29 kDa, alcohol dehydrogenase 150 kDa, β-amylase 200 kDa, BSA 66 kDa, Sigma-Aldrich, Inc., St. Louis, MO, USA) or by calibrating the light scattering detector using the monomeric peak of BSA and light-scattering intensity (SLS).
3
Purification of Hydrolyzed Peptides Using Preparative HPLC
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Hydrolyzed peptides, using Alcalase®, were purified using an Xbridge BEH preparative C18 5 μm OBD 19 × 250 mm (Waters) connected to a Shimadzu Prominence LC-20A system, including a CBM-20A controller, two LC-20 AP preparative pumps and a DGU-20A3R online degasser. An SPD-20A UV with a preparative cell (0.5 mm) was used as a detector and was set at 214nm. An FRC-10A Shimadzu was employed as an auto collector. Data acquisition was performed by the LabSolution version 5.53 software (Shimadzu, Kyoto, Japan).
The sample was eluted with a flow rate of 17 mL min−1 using ddH2O/TFA (99.9/0.1, v/v) as phase A and MeOH/TFA (99.9/0.1, v/v) as phase B. The gradient started with 1 min of 25% phase B, and then increased to 50% in 19 min; finally, B was increased to 95% in 1 min and maintained constant for 5 min. The column was re-equilibrated for 6 min at 25% B. Ten fractions were collected every 3 min (except for fraction 1 and 12, as shown inFigure 2 ). Collected peptide fractions (F1–F10) were subjected to bioactivity tests to identify the most active ones.
The sample was eluted with a flow rate of 17 mL min−1 using ddH2O/TFA (99.9/0.1, v/v) as phase A and MeOH/TFA (99.9/0.1, v/v) as phase B. The gradient started with 1 min of 25% phase B, and then increased to 50% in 19 min; finally, B was increased to 95% in 1 min and maintained constant for 5 min. The column was re-equilibrated for 6 min at 25% B. Ten fractions were collected every 3 min (except for fraction 1 and 12, as shown in
4
Preparative HPLC Purification of Phenolic Compounds
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Extracted phenolic compounds were purified using an Xbridge BEH preparative C18 5 μm OBD 10 × 250 mm (Waters) connected to Shimadzu Prominence LC-20A system, including a CBM-20A controller, two LC-20 AP preparative pumps, a DGU-20A3R online degasser. An SPD-20A UV with a preparative cell (0.5 mm) was used as detector. A FRC-10A Shimadzu was employed as the autocollector. Data acquisition was performed by the LabSolution version 5.53 software (Shimadzu, Kyoto, Japan). The detector was set at 320 nm. Samples were eluted with a flow rate of 7 mL min−1 using ddH2O/TFA (99.9/0.1, v/v) as mobile phase A and MeOH/TFA (99.9/0.1, v/v) as mobile phase B. The gradient started from 0% B and then increased to 60% in 25 min; then, B was increased to 100% and maintained constant for 4 min. The column was re-equilibrated for 2 min. Ten fractions were collected every 3 minutes, except for fraction 1 and 2 (dead volume) and 9 and 10 (washing and column equilibration). Collected fractions (F3–8) were analyzed by UHPLC coupled to high resolution mass spectrometry.
5
Purification and Bioactivity Screening of Phenolic Compounds
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Phenolic compounds were purified using a column Xbridge® BEH C18 (4.6 mm × 250 mm, 130 Å, 5µm particle size, Waters, Milford, MA, USA). The column was connected to the Shimadzu Prominence LC-20AD system, including a CBM-20A controller, two LC-20 AD XR pumps, and a DGU-20As online degasser, equipped with an SPD-M20A UV detector, and an autocollector FRC-10A (Shimadzu) was employed. Data acquisition was performed by the LabSolution version 5.53 software (Shimadzu, Kyoto, Japan). The detector was set at 280 nm. The sample was eluted at a flow rate of 0.6 mL min−1 using ddH2O with 10 mmol L−1 ammonium formate at pH 10 as phase A and MeOH:ddH2O (90:10, v/v) with 10 mmol L−1 ammonium formate at pH 10 as phase B. The gradient started at 5% of B, then increased to 50% in 44 min; then, the column was equilibrated for 10 min. Six fractions were collected as follows: F1 (1–11 min), F2 (12–14 min), F3 (15–16 min), F4 (16–19 min), F5 (20–25 min), and F6 (26–40 min). The number of collected fractions from chromatographic separation is shown in Figure 1 A.
Each collected fraction (F1–6) was subjected to a PLpro bioactivity test to identify the most active ones.
Each collected fraction (F1–6) was subjected to a PLpro bioactivity test to identify the most active ones.
6
SEC Separation of Peptide Hydrolysate
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The total hydrolysate was subjected to peptide separation using a BIOBASIC SEC 120, 5 μm, 150 x 7.8 mm (Thermo, Waltham, Massachusetts, USA) connected to a Shimadzu Prominence LC-20A system, including a CBM-20A controller, two LC-20 AP preparative pumps and a DGU-20A3R online degasser. An SPD-20A UV with a preparative cell (0.5 mm) was used as a detector and was set at 214nm. An FRC-10A Shimadzu was employed as an auto collector. Data acquisition was performed by the LabSolution version 5.53 software (Shimadzu, Kyoto, Japan).
The sample was eluted in isocratic with a flow rate of 1 mL/min using ddH2O/TFA (99.9/0.1, v/v).
Two fractions were collected, as shown in Figure 1S. The fraction collected from 1 to 5 min contained medium-sized peptides, while the fraction collected from 6 to 10 min contained short peptides.
The dry weight of the mixture was 14.7 mg (98% w/w).
The sample was eluted in isocratic with a flow rate of 1 mL/min using ddH2O/TFA (99.9/0.1, v/v).
Two fractions were collected, as shown in Figure 1S. The fraction collected from 1 to 5 min contained medium-sized peptides, while the fraction collected from 6 to 10 min contained short peptides.
The dry weight of the mixture was 14.7 mg (98% w/w).
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