Proteins were incubated for 10 min at room temperature in the presence of the indicated effectors and injected on a Superdex 200 PC3.2/30 column mounted on an Ettan LC system (GE Healthcare) run at room temperature and at a 40 μl/min flow rate.
Ettan lc system
Manufactured by GE Healthcare
Sourced in United States, Norway
The Ettan LC system is a modular HPLC instrument designed for high-performance liquid chromatography. It features a low-pressure gradient pump, an autosampler, and a variable wavelength UV/Vis detector. The Ettan LC system is capable of performing analytical and preparative scale chromatographic separations.
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Lab products found in correlation
Superdex 200 increase 10 300 column, by GE Healthcare (2 mentions)
Dawn heleos 2, by Wyatt Technology (2 mentions)
Optilab rex, by Wyatt Technology (2 mentions)
Superdex 200 pc 3.2 30, by GE Healthcare (2 mentions)
Superdex 200 pc 3.2 30 column, by GE Healthcare (1 mentions)
Itraq 4plex, by AB Sciex (1 mentions)
Radiomatic 610tr flow system analyzer, by PerkinElmer (1 mentions)
Jupiter c18 rp column, by Phenomenex (1 mentions)
A4416, by Merck Group (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Cell disrupter, by Constant Systems (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Superose 6 increase 3.2 300 mm column, by GE Healthcare (1 mentions)
Seppak cartridge, by Waters Corporation (1 mentions)
Sep pak cartridges, by Waters Corporation (1 mentions)
Tmt six plex reagents, by Thermo Fisher Scientific (1 mentions)
12 protocols using ettan lc system
1
Protein Separation by Size Exclusion
2
Multiplexed Protein Quantification by iTRAQ
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Isobaric tags for relative and absolute quantification (iTRAQ) analysis
was performed using 10 μg of in-solution digested sample from
each patient and control. Samples were labeled with iTRAQ 4plex (AB
SCIEX, Framingham, MA, USA) according to the manufacturer’s
protocol, resulting in a total of five sets of 4plex experiments (Figure1 ). A mixed sample containing 5 μg of sample
from each of the patients and controls was labeled with the iTRAQ
114-tag in all five experiments and used to normalize between experiments.
Each 4plex experiment was diluted 10 times in buffer A (0.5% formic
acid and 5% acetonitrile) and separated on a Higgins Analytical strong
cation exchange (SCX) column (PL-SCX 1000 Å 5 μm 20 ×
2.1 mm column, Higgins Analytical, Rengstorff, CA, USA) equilibrated
in buffer A and connected to an Ettan LC system (GE Healthcare, Wauwatosa,
WI, USA). The peptides were eluted using a 100 μL/min flow rate
and a 1% B/min linear gradient from buffer A to buffer B (buffer B
containing 1 M NaCl). The 15 collected fractions were lyophilized,
redissolved in 100 μL 0.1% formic acid, and desalted using POROS
50 R2 material packed in GELoader tips. The desalted samples were
lyophilized and resuspended in 12 μL 0.1% formic acid and stored
at −20 °C.
was performed using 10 μg of in-solution digested sample from
each patient and control. Samples were labeled with iTRAQ 4plex (AB
SCIEX, Framingham, MA, USA) according to the manufacturer’s
protocol, resulting in a total of five sets of 4plex experiments (Figure
from each of the patients and controls was labeled with the iTRAQ
114-tag in all five experiments and used to normalize between experiments.
Each 4plex experiment was diluted 10 times in buffer A (0.5% formic
acid and 5% acetonitrile) and separated on a Higgins Analytical strong
cation exchange (SCX) column (PL-SCX 1000 Å 5 μm 20 ×
2.1 mm column, Higgins Analytical, Rengstorff, CA, USA) equilibrated
in buffer A and connected to an Ettan LC system (GE Healthcare, Wauwatosa,
WI, USA). The peptides were eluted using a 100 μL/min flow rate
and a 1% B/min linear gradient from buffer A to buffer B (buffer B
containing 1 M NaCl). The 15 collected fractions were lyophilized,
redissolved in 100 μL 0.1% formic acid, and desalted using POROS
50 R2 material packed in GELoader tips. The desalted samples were
lyophilized and resuspended in 12 μL 0.1% formic acid and stored
at −20 °C.
3
Radio-Labeling of Cypep-1 using Iodo-Gen
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The radio labelling of Cypep-1 was performed with 125I, according to the Iodo-Gen method as described by Fraker et al. [83 (link)]. The obtained 125I-Cypep-1 stock solution was stored in the dark at 4°C for 48 hours prior to validation by reversed phase chromatography, using a 4.6 × 50 mm Dionex Proswift RP-2H column on an Ettan LC System (GE Healthcare, Oslo, Norway) in line with a Radiomatic 610TR Flow System Analyzer by Perkin Elmer (PerkinElmer, Waltham, MA) for gamma detection.
4
SEC-MALLS Analysis of Protein Samples
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SEC-MALLS data were collected using an Ettan LC system (GE Healthcare) with a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated in buffer E (20 mM Tris HCL, pH 8.0, 80 mM NaCl, and 0.1 mM TCEP) at a flow rate of 0.5 ml min−1. The system was coupled on-line to an 18-angle MALLS detector and a differential refractometer (DAWN HELEOS II and Optilab rEX, Wyatt Technology). Molar mass determination was calculated with ASTRA 6.2 software.
5
HPLC Analysis of Disaccharide Profiles
6
Superdex 200 Chromatography of Proteins
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Proteins were preincubated for 15 min at 20°C and injected on a superdex 200 PC 3.2/30 (GE Healthcare) mounted on an EttanLC system (GE Healthcare) and run at 20°C. Unless noted otherwise, preincubation and equilibration buffers were: 55 mM Tris–HCl (pH 8.0), 1.7% sucrose, 33 mM KI, 24 mM tripotassium citrate, 9 mM Mg acetate, 0.09 mM EDTA, 0.14 mM ATP, 10 μM pyridoxal 5′-phosphate and 50 mM Tris–HCl (pH 8.0) containing 33 mM tripotassium citrate, 10 mM Mg acetate, 0.1 mM EDTA, 0.1 mM ATP, 10 μM pyridoxal 5′-phosphate, respectively. Buffers were supplemented with maltotriose and DTT when indicated. The column was calibrated with three standard globular proteins: thyroglobuline (669 kDa), β-amylase (200 kDa) and BSA (66 kDa).
7
SEC-MALLS Protein Analysis Protocol
8
Gel Filtration of Cellular Extracts
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Gel filtration of cellular extracts from stationary phase cultures of strains VF7969 and VF9849 was performed as previously described (20 (link)). Crude cell extracts were obtained using a Cell Disrupter (Constant Systems, Daventry, UK) in 10-mM Tris-HCl pH 7.9, 0.1-mM DTT, 0.1-mM ethylenediaminetetraacetic acid, glycerol 5%, 300-mM NaCl supplemented with antiprotease (Roche). A total of 20 μl of the supernatant was applied to a gel filtration column (Superdex 200 PC3.2/30, GE Healthcare) using the EttanLC System (GE Healthcare). Elution was performed at a flow rate of 0.04 ml/min at room temperature, gathering fractions of 50 μl. The proteins in the elution fractions were analyzed by SDS-PAGE and electroblotted onto nitrocellulose membranes (Hybond ECL GE Healthcare). Immunoblot analysis of the eluted fractions was carried out using the σS rabbit antibody (47 (link)) as described above and monoclonal antibodies against the β′ and α subunits of RNAP (WP001 and WP003, Neoclone) and a secondary anti-mouse antibody linked to peroxidase (A4416, Sigma-Aldrich).
9
Quantitative Analysis of Disaccharide Conjugates
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The disaccharide 2-sulfoglucuronate-N-acetyl-glucosamine (G2A0) was pre-labelled with the fluorescent dye 2-aminoacridone (AMAC).10 (link) AMAC-labelled G2A0 (12,5 nmol) was incubated with 100 µg protein of the appropriate homogenates in a final volume of 52,5 µl in 250 mM ammonium-acetate buffer pH 4.6 for 24 hours at 37°C. After centrifugation (15000 g, 4°C), the samples were analysed by C18-reversed-phase (RP)-chromatography in ammonium acetate buffer (60 mM, pH 5.6) with a flow rate of 1 mL/min with the Ettan LC system (GE Healthcare). The saccharides were eluted and fractionated with an acetonitrile gradient, in which AMAC-labelled disaccharides were detected by ultraviolet (UV) absorbance at 255 nm. The fractions containing AMAC-mediated fluorescence peaks were vacuum dried, resolved in 25 µl acetonitrile and analysed by nano-electrospray ionization mass spectrometry (ESI-MS) in a negative ion mode.
10
Analytical Size Exclusion Chromatography
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Analytical size exclusion chromatography was performed over a Superdex 200 3.2/300 increase column equilibrated with 50 mM HEPES-NaOH pH 7.5, 50 mM KCl using an Ettan LC system (GE Healthcare Life Sciences). Expected molecular masses were calculated based on the separation of the gel filtration standard (Bio-Rad) by the column.
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