Salts were purchased from Sigma-Aldrich (Milan, Italy). Reagents were obtained from Mallinckrodt Baker (Milan, Italy). The Sartorius Vivaflow 200 ultrafiltration system, equipped with a 10 kDa cutoff polythersulfone membrane, was obtained from Sartorius (Florence, Italy). Pierce Concentrators were from Thermo Fisher Scientific (Milan, Italy). Anion exchange chromatography was performed on an FPLC Akta Basic equipped with a UV-Vis detector (GE-Healthcare, Milan, Italy) using a HiTrap DEAE FF column with a volume of 5 mL (GE-Healthcare, Milan, Italy). Gel filtration chromatography was conducted on an HPLC Akta Basic equipped with a UV-Vis detector (GE Healthcare, Milan, Italy) using a progel-TSK G2000 SWXL column 30 cm × 7.8 mm (Supelco, Merck KGaA, Darmstadt, Germany). Dialysis was performed using Spectra/Por 3.5 kDa MWCO membranes (Spectrum Labs, Fisher Scientific Italia, Rodano, Milan). PVDF membranes for western blotting were obtained from Millipore (Milan, Italy). The monoclonal antibody mAbKT4 was used to detect the KT [9 (link)]. The anti-mouse secondary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The ECL (enhanced chemiluminescence) system used for the immunodetection was obtained from Amersham Pharmacia Biotech (Milan, Italy).
Pierce concentrator
Manufactured by Thermo Fisher Scientific
Sourced in United States
Pierce Concentrators are compact, single-use devices designed to concentrate and desalt samples. They operate using centrifugal force to reduce sample volume while retaining the desired proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (2 mentions)
Hitrap deae ff column, by GE Healthcare (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Sephadex g 25 column, by GE Healthcare (1 mentions)
Histrap hp 5 ml column, by GE Healthcare (1 mentions)
Akta pure fplc system, by GE Healthcare (1 mentions)
Akta pure, by Cytiva (1 mentions)
Papain solution, by Merck Group (1 mentions)
Hiload 16 60 superdex 200, by GE Healthcare (1 mentions)
Immobilized anti tmt resin, by Thermo Fisher Scientific (1 mentions)
Pierce s nitrosylation western blot kit, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Usp2 catalytic domain, by R&D Systems (1 mentions)
9 protocols using pierce concentrator
1
Purification and Detection of KT Protein
2
Radiolabeling and Pharmacokinetics of scFv7F9 Constructs
3
Purification of Recombinant Bteqβgluc
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The chromatograms for purification of Bteqβgluc are presented in the supplementary files (Fig. S2 ). Recombinant Bteqβgluc produced in E. coli was purified by immobilized metal affinity chromatography (IMAC) using a HisTrap HP 5 ml column connected to an AKTA Pure FPLC system (GE Healthcare, Sweden). The mobile phase was 50 mM Tris–HCl (pH 7.5), 300 mM NaCl and 20 mM imidazole with a flow rate of five mL/min. Elution was performed on a gradient of imidazole (20 to 500 mM over 10 column volumes) at a flow rate of 3 mL/min.
Anion exchange chromatography was used for purification of recombinant Bteqβgluc produced in yeast cultures. Protein samples were loaded on a HiTrap Q-FF column (five mL) pre-equilibrated in 50 mM Tris–HCl (pH 7.5), and elution was performed on a linear gradient of NaCl (0–1 M, 10 column volumes) at a flow rate of 3 mL/min.
Purified Bteqβgluc was concentrated and desalted against Milli Q water using Pierce Concentrators (9K MWCO, Thermo Fisher Scientific, USA) at 4 °C. Samples were then analyzed on 10% SDS-PAGE and by Western blotting as detailed below.
Anion exchange chromatography was used for purification of recombinant Bteqβgluc produced in yeast cultures. Protein samples were loaded on a HiTrap Q-FF column (five mL) pre-equilibrated in 50 mM Tris–HCl (pH 7.5), and elution was performed on a linear gradient of NaCl (0–1 M, 10 column volumes) at a flow rate of 3 mL/min.
Purified Bteqβgluc was concentrated and desalted against Milli Q water using Pierce Concentrators (9K MWCO, Thermo Fisher Scientific, USA) at 4 °C. Samples were then analyzed on 10% SDS-PAGE and by Western blotting as detailed below.
4
Production and Concentration of Soluble Toxins
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The strains WaF17.12, WaATCC 96603 and WaUM3 were grown in culture conditions suitable to stimulate the production of soluble toxins in liquid medium, as reported by İzgü et al. (2006) [33] : YPD broth (20 g/l peptone, 20 g/l glucose, 10 g/l yeast extract), buffered at pH 4.5 or pH 8 with 0.1 M citric acid and 0.2 M K2HPO4, and incubated at 26°C for 36 h at 70 rpm [32] (link). The yeast cells were removed by centrifugation at 2000 g 4°C for 10 min. The supernatants were filtered and concentrated 100-fold using Pierce Concentrators (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) with a cut off of 9 kDa, by centrifugation at 4°C for 5 h at 2000 g.
5
Purification of Photosystem I Complexes
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Thylakoid membranes were prepared from cells in late exponential growth phase as described in ref. (Sun et al. 1999) (link). To purify the PS I complexes from the thylakoid membranes, the latter concentration was adjusted to 0.5 mg of Chl per mL in 10 mM MOPS-NaOH, pH 7.0 buffer and the membranes were solubilized for 2 h at 4 °C by adding 1% n-dodecyl-Dmaltopyranoside (DM). The solution was centrifuged for 20 min at 15,000 x g to remove insoluble debris, the supernatant was loaded onto a 10-30% (w/v) linear sucrose gradient prepared in 10 mM MOPS-NaOH pH 7.0 buffer containing 0.05% DM and ultracentrifuged for 16-18 h at 150,000 x g at 4 °C. The lower dark green band containing the PS I trimers was carefully collected and concentrated by using Pierce concentrators (20,000 molecular weight cut-off membranes, Thermo Scientific) to a final concentration of 1.0 μg/μL Chl. When needed, the samples were stored at -80 °C for further use.
6
BRCA1-BARD1 DUB Assay Protocol
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BRCA15382insC-BARD1 protein fractions isolated from H2O2-treated cells were used for DUB assays. The reaction mixture (total volume, 200 μl) contained 180 μl of the protein fraction and 20 μl of 10× USP2 catalytic domain (final concentration, 500 nM; UbiCREST, K-400; Boston Biochem). Control mixtures were prepared with 180 μl of the protein fraction and 20 μl of 1× DUB reaction buffer (UbiCREST, K-400; Boston Biochem) lacking the enzyme. Both reaction and control tubes were incubated in a water bath at 37°C. After 30 min, samples were directly analyzed by either EM imaging or SDS-PAGE and Western blot analysis. Before EM specimen preparation, free ubiquitin was removed from the samples using Pierce Concentrator (100K MWCO, 0.5 ml; Thermo Fisher Scientific).
7
Papain Digestion and Fab Purification
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A2C7 (mouse IgG2b), A27D7, and A20G2 (mouse IgG2a) Mabs were digested with 2% (wt/wt) activated papain for 4hr at 37°C in digestion buffer (A27D7: 50 mM NaOAc pH 5.5; A2C7 and A20G2: PBS pH 7.4). Papain was activated by incubating 24.4 μL papain solution at 20.5 mg/mL (Sigma) in 1 mL solution containing 100 μL 10X Papain Buffer (1M NaOAc pH 5.5, 12mM EDTA, and 10mM cysteine) for 15 min at 37°C. The papain digestion was inhibited with 1 mL of 200 mM iodoacetamide (IAA). A27D7 sample was then dialyzed in 2 L PBS pH 8.0 overnight at 4°C. For A2C7 and A20G2, papain reaction mixtures were diluted in one volume of PBS pH 8.0. All three samples were passed through a pre-equilibrated 1 mL protein A FF column in PBS pH 8.0 binding buffer. The purified Fab, contained in the protein A flowthrough, was concentrated to 1 mL using centrifugal filtration devices (Pierce Concentrator; 9-kDa molecular mass cut-off (MWCO)–Thermo Scientific) and purified to homogeneity by Size Exclusion Chromatography (SEC) on a Superdex 200 HiLoad 16/60 (GE) column in 20mM Tris pH 8.0, 200mM NaCl running buffer. All Fabs eluted as a single and sharp peak (Ve ~ 86 mL).
8
Detecting S-Nitrosylated Proteins via Biotin-Switch Assay
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The biotin-switch assay (BST) is a modified immunoblot analysis used to detect the levels of S-nitrosylated proteins. Here, we used a modified version of this assay. In brief, cell lysates were prepared from A-Cre– or A-GFP–treated mdx;STOPFloxBmi1 satellite cell cultures in HENS Buffer (Pierce S-Nitrosylation Western Blot kit; Thermo Fisher Scientific) and the BCA Protein Assay was used (Thermo Fisher Scientific) to determine the protein concentration. 300 µg of protein from each sample was mixed with 20 mM methyl methane thiosulfonate (MMTS) for 30 min at room temperature to block free thiol groups. After removing excess MMTS by acetone precipitation (1 h at −20°C), S-nitrosothiols were reduced to thiols with 20 mM sodium ascorbate. In the negative control, ultrapure water was used instead of sodium ascorbate. Labeling reagent containing tandem mass tag (TMT) was added to label the newly formed thiols. After acetone precipitation, samples were resuspended into HENS buffer. TMT labeled proteins were loaded onto spin columns with pre-added immobilized anti-TMT resin (Thermo Fisher Scientific) and eluted. The eluted proteins were concentrated using Pierce Concentrator (Thermo Fisher Scientific) and further analyzed by immunoblotting. Input was used for the control blot.
9
Deubiquitination of BRCA1 by USP2
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