Freeze-dried alkali fractions (2 mg) were dissolved in ammonia solution (w = 30%) and heated (60°C, 1 h) to solubilize the extracts. HPSEC was performed on a Dionex (Sunnyvale, USA) Ultimate 3000 UPLC system equipped with three TSKgel SuperAW columns (6.0 mm ID × 150 mm per column; 4 μm) in series (SuperAW4000, SuperAW3000, SuperAW2500; Tosoh Bioscience, Stuttgart, Germany), in combination with a guard column (Tosoh Bioscience, Stuttgart, Germany). Columns were eluted at 40°C with 0.2 mol/L sodium nitrate at 0.6 mL/min. The eluate was monitored with a Shodex RI-101 (Kawasaki, Japan) refractive index (RI) detector. Dextran standards (Sigma-Aldrich, Poole, Dorset, UK) were used for Mw calibration.
Guard column
Manufactured by Tosoh
Sourced in Japan, Germany, United States
A guard column is a short, narrow-bore chromatography column placed before the analytical column in a liquid chromatography system. Its primary function is to protect the analytical column from contaminants and particulates in the sample, extending the column's lifetime and performance.
Automatically generated - may contain errors
Lab products found in correlation
Nexera xr uhplc system, by Shimadzu (2 mentions)
Tskgel supersw3000, by Tosoh (2 mentions)
Dextran standard, by Merck Group (1 mentions)
Superaw4000, by Tosoh (1 mentions)
Superaw3000, by Tosoh (1 mentions)
Superaw2500, by Tosoh (1 mentions)
Sw2000, by Tosoh (1 mentions)
Tskgel g2000sw, by Tosoh (1 mentions)
E2695 hplc, by Waters Corporation (1 mentions)
Gel filtration molecular weight standard, by Bio-Rad (1 mentions)
Lc solution software, by Shimadzu (1 mentions)
Tskgel g4000swxl column, by Tosoh (1 mentions)
Tskgel bioassist g3swxl column, by Tosoh (1 mentions)
Nexera xr hplc system, by Shimadzu (1 mentions)
Super sw3000, by Tosoh (1 mentions)
Spd m20a, by Shimadzu (1 mentions)
Tskgel g2000swxl, by Tosoh (1 mentions)
10 protocols using guard column
1
High-Performance Size-Exclusion Chromatography of Alkali Extracts
2
Size-exclusion chromatography analysis
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Size-exclusion chromatography (SEC) analysis was performed using TSKgel SuperSW 3000 (4.6 mm × 30 cm, 5 μm) with a guard column (Tosoh Bioscience, Inc., South San Francisco, CA, USA) equipped on the Nexera XR UHPLC system (Shimadzu Co., Kyoto, Japan) as previously reported as the standard procedures [21 (link)].
3
Neuropeptide Y Fractionation by Triple-GFC
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Three GFC columns, TSKgel Super SW3000, SW2000, and SW2000 (4.6 × 300 mm; 4 μm) with a guard column (TOSOH Corporation, Tokyo, Japan), were connected in series as a triple‐analytical GFC column. The eluent was an aqueous solution of 40% acetonitrile/0.1%TFA, and a flow rate of 0.15 ml/min was used. An aliquot of 100‐ml sample of large‐scale GFC was loaded to the triple‐analytical GFC column. The eluted fractions were taken to each tube systematically for two mins. NPY samples were eluted in 54–66 min for six fractions of Fr.1–Fr.6 as shown in Figure 4 . These fractions were dried‐up with a centrifugal evaporator system (EYELA Tokyo Rikakikai Co. Ltd., Tokyo, Japan), and then, they were resolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution for nanoLC‐orbitrap MS analysis.
4
Purification and Analysis of NPYs
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The GFC column of TSKgel G2000SW (7.5 × 600 mm; 10 μm) with a guard column (TOSOH Corporation, Tokyo, Japan) was used as the first column. The eluent was an aqueous solution of 40% acetonitrile/0.1%TFA, and a flow rate of 1 ml/min was used. The sample fractions were collected at intervals of 1 min. The lyophilized peptide extract (2.9 mg) was dissolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution, and all of them were loaded to the GFC column. The eluted fractions were taken to each tube systematically for 1 min. The NPYs were eluted at the 18 min fraction as shown in Figure 4 , which was dried up by a centrifugal evaporator system (EYELA Tokyo Rikakikai Co. Ltd., Tokyo, Japan), and it was dissolved in 100 μl of 40% acetonitrile/0.1%TFA aqueous solution for the subsequent triple analytical GFC.
5
RP-HPLC Purification of Recombinant Caspase-2
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Experiments were performed on a Tosoh TSKgel Protein C4-300, L × I.D. 5 cm × 4.6 mm, 3 μm column with a guard column (Tosoh, Tokyo, Japan) on a Waters e2695 HPLC (Waters, Milford, MA, USA). Mobile phase A was water with 0.15% trifluoroacetic acid (TFA) and mobile phase B was acetonitrile with 0.15% TFA. The flowrate was 1 mL/min. Temperature of the column oven was 40 °C, temperature of the autosampler 10 °C. After a 2-min wash, a gradient from 25–50% B in 6 min, followed by a gradient from 50–55% B in 7 min was used to separate cpCasp2 from host cell proteins.
In addition, 200 µL of purified cp caspase-2 (or variant) sample (~4 g/L) was diluted with 100 µL PBS and 100 µL 2 M dithiothreitol (DTT). Furthermore, 10 µl of 0.22 µm filtered sample were injected. The outlet was monitored at 214 nm and 280 nm. The HCP peaks eluted between retention times 3.8 and 9 min. The cpCasp2 peaks eluted between 9.2 and 12.4 min. The peak areas in the 214 nm signal were used to calculate the purity of cpCasp2.
In addition, 200 µL of purified cp caspase-2 (or variant) sample (~4 g/L) was diluted with 100 µL PBS and 100 µL 2 M dithiothreitol (DTT). Furthermore, 10 µl of 0.22 µm filtered sample were injected. The outlet was monitored at 214 nm and 280 nm. The HCP peaks eluted between retention times 3.8 and 9 min. The cpCasp2 peaks eluted between 9.2 and 12.4 min. The peak areas in the 214 nm signal were used to calculate the purity of cpCasp2.
6
SEC Analysis of Protein Samples
7
Size-Exclusion HPLC Analysis of Antibodies
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A Shimadzu Prominence ultrafast liquid chromatography HPLC system equipped with a diode array detector (with absorbance detection at 214 nm) was used. The system was equilibrated at 0.5 mL/min flow rate in 0.2 M sodium phosphate buffer at pH 6.8 for at least 2 h. Ten μL of each Ig (10 μg total protein) was injected and separated by a TOSOH TSKgel G4000SWXL column (8 μm particle size, 7.8 mm ID × 30 cm) for sIgA or a TOSOH TSK-Gel BioAssist G3SWxl column (5 μm size, 7.8 mm ID × 30 cm) for IgG1 with a corresponding guard column operated at ambient temperature (Tosoh Biosciences) using a 30-min run time. Gel filtration molecular weight standards (Bio-Rad, Hercules, CA) were injected before and after the Ig sample sets to ensure integrity of the column and HPLC system. Potential presence of larger aggregates was determined by running Ig samples with and without the size-exclusion chromatography (SEC) column (i.e., protein percentage recovery). Greater than 95% protein recovery was obtained for each of the 3 mAbs by SE-HPLC, indicating minimal loss of protein (e.g., larger aggregates) by using optimized SE-HPLC conditions for sIgA versus IgG1. Data were analyzed using LC-Solution software (Shimadzu, Kyoto, Japan).
8
Quantifying Tenecteplase One- and Two-Chain Forms
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High-performance size-exclusion chromatography (HP-SEC) was used to quantify the one- and two-chain forms of tenecteplase. After reducing the disulfide bonds with dithiothreitol (DTT), the one-chain material was separated from the two-chain material based on its larger molecular weight and size. Analysis was monitored at 210 nm using a size-exclusion chromatography (SEC) column with a guard column (Tosoh Bioscience, Stuttgart, Germany) and sodium dodecyl sulfate (SDS)/sodium phosphate buffer as the mobile phase.
9
SEC Analysis of Protein Samples
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SEC
analysis was performed
on a Nexera XR HPLC system (Shimadzu Co., Kyoto, Japan). Approximately
25 μg of protein was loaded onto a TSKgel SuperSW 3000 (4.6
mm × 30 cm, 5 μm) with a guard column (Tosoh Bioscience,
Inc., South San Francisco, CA, USA) and eluted using an isocratic
flow (37 min, 0.25 mL/min) of 200 mM sodium phosphate with 10% of
acetonitrile at pH 6.8. The absorption of the elution was monitored
at wavelengths of 280 and 700 nm.
analysis was performed
on a Nexera XR HPLC system (Shimadzu Co., Kyoto, Japan). Approximately
25 μg of protein was loaded onto a TSKgel SuperSW 3000 (4.6
mm × 30 cm, 5 μm) with a guard column (Tosoh Bioscience,
Inc., South San Francisco, CA, USA) and eluted using an isocratic
flow (37 min, 0.25 mL/min) of 200 mM sodium phosphate with 10% of
acetonitrile at pH 6.8. The absorption of the elution was monitored
at wavelengths of 280 and 700 nm.
10
HPSEC analysis of NGO-MIX and NGO (3.5-10K)
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The HPSEC of NGO-MIX and NGO (3.5-10K) was conducted on a HPLC system with multi-wavelength absorbance detector SPD-M20A (Shimadzu, Japan), operational in the 200-500 nm range. The fluorescence detector RF-20A, connected directly to the line of the absorbance detector, was set to an excitation wavelength 270 nm, emission wavelength 450 nm, and used for measurements of fluorescence emission. The column TSKgel G2000SW XL (7.8 mm ID, 300 mm L, particles size 5.0 µm, average pore size 12.5 nm) equipped with a guard column (Tosoh Bioscience, Japan) with a fractionation range from 150 to 5 kDa for globular proteins was used. The 30 mM phosphate buffer (pH 6.5) was used as eluent. The flow rate was set to 1.0 ml min -1 . The void column volume (V o ) and the permeation volume (V p ) were 5.6 and 13.1 ml, respectively. The solutions of NGO-MIX and NGO (3.5-10K) applied on the column had an absorbance of 1.0 at 270 nm in 30 mM phosphate buffer, and the volume of injection was 0.02 ml. The phosphate buffer used as the mobile phase did not show any absorbance or fluorescence peaks. The entire HPSEC procedure was repeated three times and the deviations did not exceed 3%. The absorbance spectra of chromatographic peaks #1-8 were extracted from the data of the multi-wavelength absorbance detector.
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