For dialysis, 10 kDa cut-off Dialysis Cassettes (Slide-A-Lyzer, ThermoScientific) were used. For proteins eluted in native conditions, dialysis was done with (Tris-HCl 20 mM pH 8, Glycerol 10%, Tween 0.05%, NaCl 250 mM, DTT 0.5 mM) at 4°C. After 1 h, buffer was changed and dialysis performed overnight (12 h). For proteins eluted in denaturing conditions, seven successive dialysis steps were performed at 4°C, with 10 kDa cut-off Dialysis Cassettes (Slide-A-Lyzer, ThermoScientific). HducCDTs were reconstituted by co-dialysis at equimolar concentrations. The first four baths used Refolding Buffer with decreasing Urea and L-Arginine concentration (Tris-HCl 100 mM pH 7.5, L-Arginine 400 mM, Glycerol 20%, Urea 5 M or 3 M or 1.5 M for baths 1 to 3, Tris-HCl 100 mM pH 7.5, L-Arginine 200 mM, Glycerol 20% for bath 4) and were changed after 24 h. The proteins were then dialyzed three times with Tris-HCl 100 mM pH 7.5, Glycerol 10% during 3 h. Finally, the protein solution was centrifuged (4°C, 13 000 g, 5 min) to remove any aggregate, snap-frozen and stored (-80°C).
Slide a lyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Slide-A-Lyzer is a dialysis device designed for the purification and buffer exchange of proteins, peptides, and other macromolecules. It facilitates the efficient removal of small molecules, salts, or other contaminants from sample solutions through the process of dialysis.
Automatically generated - may contain errors
Lab products found in correlation
Ni nta superflow cartridge, by Qiagen (5 mentions)
Sfca filter, by Thermo Fisher Scientific (5 mentions)
Fluorescence activated cell sorting (facs), by Merck Group (5 mentions)
Amicon ultra, by Merck Group (3 mentions)
Protease inhibitor, by Roche (3 mentions)
3500 mwco, by Thermo Fisher Scientific (2 mentions)
Sw41ti rotor, by Beckman Coulter (2 mentions)
Infinite m200 pro, by Tecan (2 mentions)
Lysozyme from chicken egg white, by Merck Group (2 mentions)
Phelper plasmid, by Cell Biolabs (2 mentions)
Paav dj plasmid, by Cell Biolabs (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Brij 35, by Thermo Fisher Scientific (2 mentions)
Igg cocktail, by Roche (2 mentions)
Ez link sulfo nhs lc biotin kit, by Thermo Fisher Scientific (2 mentions)
Itc200, by Malvern Panalytical (1 mentions)
10k mwco, by Thermo Fisher Scientific (1 mentions)
131 protocols using slide a lyzer
1
Optimized Dialysis and Refolding of Recombinant Proteins
2
Nanoprecipitation Procedure for PLA-PEG Nanoparticles
3
Thermodynamic analysis of PXR-ligand interactions
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Purified PXR(130-434)-SRC-1 was dialysed overnight against Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM using 10 kDa molecular weight cut-off dialysis cassettes (Slide-A-Lyzer 0.5 ml 10 K MWCO, Thermo Scientific). Protein concentration was determined spectrophotometrically (ɛ280 nm=26,210 l mol−1 cm−1). Duplicate experiments were performed on Microcal ITC200 (Malvern) operating at 25 °C. Titrations were carried out in Tris-HCl 20 mM, pH 8.5, NaCl 200 mM, TCEP 1 mM supplemented with 0.05% Tween 20 and 5% DMSO (syringe, sample and reference cells). PXR (5 μM) was disposed in 200 μl cell and compounds were delivered from 40 μl syringe. Compound solutions were set to 300 μM when tested individually (Fig. 5b,c ), 50 μM each when used simultaneously (Fig. 5f ) and 50 μM (EE2, Fig. 5d ) or 200 μM (TNC, Fig. 5e ) when tested after pre-incubation of PXR with 50 μM TNC or EE2, respectively. Heat exchanges were monitored throughout titrations consisting of 19 injections (one time 0.5 μl followed by 18 times 2 μl) of compound solutions into the cell containing PXR solution. Data analysis and thermodynamic parameter fitting used Microcal Origin software (Malvern).
4
Affinity Purification of 53BP1 and p53 Complexes
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Cells lyzed in Benzonase Lysis Buffer (20 mM HEPES [pH 7.9], 40 mM KCl, 2 mM MgCl2, 12% glycerol, 0.5% CHAPS, 50 U/ml Benzonase [Novagen], 0.05% [v/v] phosphatase inhibitors [Sigma-Aldrich] and protease inhibitors [Roche]) were supplemented with KCL to a 450 mM final concentration and gently mixed for 30 min at 4°C. Clarified lysates were then cassette dialyzed (Slide-A-Lyzer, Thermo Fisher Scientific) in dialysis buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 10% Glycerol, 0.5 mM DTT, 0.5 mM PMSF, 5 mM NaF, 10 mM β-glycerolphosphate). Flag-HA-53BP1 or endogenous p53 complexes were purified from 1–2 mg total protein using anti-FLAG M2 magnetic resin (Sigma-Aldrich) or p53 DO-1 antibody (Santa Cruz Biotechnology) coupled to protein G Dynabeads (Invitrogen). Protein-bead complexes washed extensively in dialysis buffer were either boiled in Laemmli buffer or eluted in 3× Flag peptide (Sigma-Aldrich).
5
Turbidity Assay for Filamentous Polymers
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Samples were dialyzed against the desired buffer in Slide-A-Lyzer mini dialysis units with 10,000 MWCO (Thermo Fisher Scientific) overnight at 4°C. Dialyzed samples were transferred to 190 μm clear-bottomed, 96-well microplates (Greiner Bio-One). Turbidity, as a value for total light scattering caused by filamentous polymers, was measured at a 350 nm wavelength with TECAN infinite M200. Unassembled FilP in 6 M urea was used as a control in all turbidity experiments because FilP did not form filaments in the presence of 6 M urea and the absorbance of completely denaturated FilP at 350 nm was zero.
6
Sortilin Expression and Purification
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Example 3
SORTECDHis was expressed in HEK-293F cells. The His-tag in the proteins enables purification with immobilized metal affinity chromatography. In this process NiNTA Superflow Cartridge (Qiagen) is equilibrated with 50 mM NAH2PO4, 300 mM NaCl and 10 mM Imidazole pH 8.0. Column is loaded with His tagged protein with a residence time of 1 minute. Column is washed with 50 mM NAH2PO4, 300 mM NaCl and 20 mM Imidazole pH 8.0. Protein is eluted with 50 mM NAH2PO4, 300 mM NaCl and 250 mM Imidazole pH 8.0. Subsequently the protein is dialyzed to PBS using a Slide-A-Lyzer with a cut off of 10.000 mwco (Thermo Scientific). After dialyzing the protein is sterile filtered using a 0.2 micron SFCA filter (Thermo Scientific).
The S18-HEK cell line was generated by transfecting HEK293 cells with a human wild type (WT) sortilin expression vector. Stable transfected cells were derived after passage in the presence of a selection agent. Individual clones were selected by dilution cloning. Clones were characterized for sortilin mRNA expression using QPCR. Highest expressing clones were than analyzed by FACS (Guava, Millipore) using an anti-sortilin polyclonal antibody (Polyclonal Goat Sortilin Biotinylated Ab, Cat. No: BAF2934 (R&D Systems)) to determine the surface expressed levels of Sortilin.
7
Sortilin Protein Expression and Purification
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Example 3
SORTECDHis was expressed in HEK-293F cells. The His-tag in the proteins enables purification with immobilized metal affinity chromatography. In this process NiNTA Superflow Cartridge (Qiagen) is equilibrated with 50 mM NAH2PO4, 300 mM NaCl and 10 mM Imidazole pH 8.0. Column is loaded with His tagged protein with a residence time of 1 minute. Column is washed with 50 mM NAH2PO4, 300 mM NaCl and 20 mM Imidazole pH 8.0. Protein is eluted with 50 mM NAH2PO4, 300 mM NaCl and 250 mM Imidazole pH 8.0. Subsequently the protein is dialyzed to PBS using a Slide-A-Lyzer with a cut off of 10.000 mwco (Thermo Scientific). After dialyzing the protein is sterile filtered using a 0.2 micron SFCA filter(Thermo Scientific).
Clones were characterized for sortilin mRNA expression using qPCR. Highest expressing clones were than analyzed by FACS (Guava, Millipore) using an anti-sortilin polyclonal antibody (Polyclonal Goat Sortilin Biotinylated Ab, Cat.No: BAF2934 (R&D Systems)) to determine the surface expressed levels of Sortilin.
8
Bioconjugation of M5A Antibody with PEG and DOTA
9
Synthesis of Au Nanoclusters using BSA
10
Protein Dialysis for SOX10 Analysis
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In a first step, the protein extract and SOX10 were subjected to dialysis. 50 μL of WCE, and 50 μL of SOX10 were pipetted in the dialysis device (Slide-A-Lyzer™, Thermofisher), and dialyzed against 1 ml of PBS, at 4 °C. After 2 h of incubation, the buffer was changed with 1 ml of fresh PBS solution for an additional overnight incubation at 4 °C.
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