SEC-MALS analysis was conducted with a high-performance liquid chromatography system (Waters, MA, USA), coupled to a ultraviolet detector, a Dawn8+ multiple-angle light scattering detector (Wyatt, CA, USA), and a refractive index detector RI500 (Shodex, Japan). Dissolved peptides were filtered through Durapore 0.1-μm centrifuge filters (Millex Sigma-Aldrich, MO, USA) and applied either to a Biosep S2000 SEC 300 × 7.8 column (Phenomenex, CA, USA) in tris buffer (50 mM tris at pH 7.5, 150 mM NaCl, and 1 mM TCEP) or to a Superdex 75 increase 10/300 column (Cytiva, MA, USA) in tris buffer supplemented with 100 μM ZnCl2. Protein samples were filtered through Durapore 0.1-μm centrifuge filters and applied either to a Superdex 75 increase 10/300 column (Cytiva, MA, USA) or to a Superdex 200 increase 10/300 column (Cytiva, MA, USA) equilibrated in Hepes buffer (50 mM Hepes at pH 8, 150 mM NaCl, and 1 mM TCEP) with or without ZnCl2. The triangular proteins were injected at a concentration of approximatively 1 mg/ml, while the proteins SBPZn1 and SBPZn2 were injected at a concentration of approximately 0.5 mg/ml. Accordingly, a different concentration of ZnCl2 was added to the mobile phase, 500 μM for triangular proteins and 20 μM for proteins SBPZn1 and SBPZn2. Analysis of the peaks of interest was conducted with Astra 7.0 software (Wyatt, CA, USA).
Superdex 75 10 300 increase column
Manufactured by Cytiva
Sourced in United States
The Superdex 75 10/300 Increase column is a gel filtration chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. It features a bed volume of 24 mL and is suitable for use with ÄKTA design chromatography systems.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 increase 10 300 column, by Cytiva (3 mentions)
Cobalt talon resin, by Takara Bio (3 mentions)
Polyethylenimine (pei), by Polysciences (3 mentions)
Expi293f suspension cells, by Thermo Fisher Scientific (3 mentions)
Superose 6 10 300 column, by Cytiva (3 mentions)
Kta pure system, by Cytiva (2 mentions)
Immobilized papain, by Thermo Fisher Scientific (2 mentions)
Astra 7, by Wyatt Technology (1 mentions)
High performance liquid chromatography system, by Waters Corporation (1 mentions)
Dawn8 multiple angle light scattering detector, by Wyatt Technology (1 mentions)
Astra software version 7, by Wyatt Technology (1 mentions)
Optilab t rex, by Wyatt Technology (1 mentions)
Dawn heleos 2 laser photometer, by Wyatt Technology (1 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (1 mentions)
Nahco3, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Ni nta column, by Qiagen (1 mentions)
Akta pure fplc, by GE Healthcare (1 mentions)
Eiger2 xe 9m, by Dectris (1 mentions)
Akta pure, by Cytiva (1 mentions)
12 protocols using superdex 75 10 300 increase column
1
SEC-MALS Analysis of Peptides and Proteins
2
Molar Mass Distribution Analysis of HIV-1 IN CTDs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Size exclusion chromatography coupled to multiangle laser light scattering (SEC-MALLS) was used to determine the molar mass distribution of the wild type and mutant HIV-1 IN CTDs. Samples (100 μL) were applied to a Superdex-75 Increase 10/300 column (Cytiva) that was equilibrated in 0.5 M NaCl, 25 mM Tris-HCl (pH 7.5), 3 mM NaN3 and 0.5 mM TCEP and was mounted on a JASCO-4000 semimicro HPLC system. Chromatography was performed at 25°C, at a flow rate of 1 mL/min. The scattered light intensities and protein concentrations of the eluted peaks were recorded using a DAWN-HELEOS II laser photometer and an OPTILAB-TrEX differential refractometer (Wyatt Technology), respectively. The weight-averaged molar mass of the material contained in the chromatographic peaks was determined using the combined data from both detectors and dn/dc = 0.186 mL/g in the ASTRA software version 7.3.2 (Wyatt Technology).
3
Spike RBD Protein Labeling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant proteins were labeled with Alexa Fluor 647 NHS ester (Thermo Fisher) as described previously (Fiedler et al., 2021 (link), 2022 (link)). In brief, solution containing 150 μg of spike RBD was mixed with dye at a three-fold molar excess in the presence of NaHCO3 (Merck) buffer at pH 8.3 and incubated at 4°C overnight. Unbound label was removed by size-exclusion chromatography (SEC) on an ÄKTA pure system (Cytiva) using a Superdex 75 Increase 10/300 column (Cytiva). Labeled and purified proteins were stored at −80°C in PBS pH 7.4 containing 10% (w/v) glycerol as cryoprotectant.
4
Purification and Amyloid Aggregation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The synthetic
wild-type Aβ42
and Aβ40 peptides were purchased from Toray Research Center,
Inc. The synthetic Aβ42 and Aβ40 peptides with a substitution
of Arg5 to Gly (R5G) or Glu (R5E) were purchased from Abclonal. The
peptides were dissolved in 6 M guanidine hydrochloride and purified
using a Superdex 75 Increase 10/300 column (Cytiva) at a 0.4 mL/min
flow rate with 20 mM sodium phosphate buffer, a pH of 7.4, to remove
from potential aggregated species. The obtained monomer was diluted
with 20 mM sodium phosphate buffer, a pH of 7.4, to the desired concentration
and supplemented with 0.1 mM ThT from a 2 mM stock solution. Then,
each sample was pipetted into multiple wells of a 96-well half-area,
low-binding polyethylene glycol coating plate with a clear bottom
(Corning 3881) at 0.1 mL per well. Aggregation assays were initiated
by placing the 96-well plate at 37 °C under quiescent conditions
in a plate reader (Infinite 200Pro, TECAN). The ThT fluorescence was
measured through the bottom of the plate with a 430 nm excitation
filter and a 485 nm emission filter. The ThT fluorescence was followed
for three repeats of each sample.
wild-type Aβ42
and Aβ40 peptides were purchased from Toray Research Center,
Inc. The synthetic Aβ42 and Aβ40 peptides with a substitution
of Arg5 to Gly (R5G) or Glu (R5E) were purchased from Abclonal. The
peptides were dissolved in 6 M guanidine hydrochloride and purified
using a Superdex 75 Increase 10/300 column (Cytiva) at a 0.4 mL/min
flow rate with 20 mM sodium phosphate buffer, a pH of 7.4, to remove
from potential aggregated species. The obtained monomer was diluted
with 20 mM sodium phosphate buffer, a pH of 7.4, to the desired concentration
and supplemented with 0.1 mM ThT from a 2 mM stock solution. Then,
each sample was pipetted into multiple wells of a 96-well half-area,
low-binding polyethylene glycol coating plate with a clear bottom
(Corning 3881) at 0.1 mL per well. Aggregation assays were initiated
by placing the 96-well plate at 37 °C under quiescent conditions
in a plate reader (Infinite 200Pro, TECAN). The ThT fluorescence was
measured through the bottom of the plate with a 430 nm excitation
filter and a 485 nm emission filter. The ThT fluorescence was followed
for three repeats of each sample.
5
Purification of Pcf1_ED Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells expressing Pcf1_ED or Pcf1_ED* with a N-terminal 6His-SUMO tag were collected by centrifugation, resuspended in lysis buffer LB6 (50 mM Tris-HCl pH8, 500 mM NaCl, 5% glycerol, 1% Triton X-100, 1 mM PMSF, 1 μM aprotinin, 0.25 mM DTT) and flash frozen in liquid nitrogen. After thawing, lysosyme was added at a final concentration of 1 mg/mL and cells were incubated 30 min at 4 °C and lysed by sonication. 6His-SUMO-Pcf1_ED was first purified on Histrap colums (Cytiva). Fractions containing the protein were pulled. SUMO protease was added at a final concentration 1/10 and the mixture was dialyzed overnight at 4 °C against the buffer DB6 (50 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM imidazole) and applied on a NiNTA column (QIAGEN) equilibrated in the DB6 buffer. The flow-through fraction containing Pcf1_ED or Pcf1_ED* was then purified by size exclusion chromatography using a Superdex 75 increase 10/300 column (Cytiva) previsouly equilibrated with the final buffer in FB6 (10 mM Tris-HCl, 50 mM HEPES pH 7, 300 mM NaCl). Fraction containing Pcf1_ED or Pcf1_ED* were concentrated using Amicon centrifuge filter units of 3 kDa cutoff (Millipore) flash freezed in liquid nitrogen and stored at –20 °C or –70 °C.
6
SEC-SAXS Structural Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SAXS was performed at BioCAT (beamline 18ID at the Advanced Photon Source, Chicago) with in-line size-exclusion chromatography (SEC-SAXS) to separate sample from aggregates and other contaminants thus ensuring optimal sample quality. The sample was loaded onto a Superdex 75 10/300 Increase column (Cytiva), which was run at 0.6 ml/min by an AKTA Pure FPLC (GE), and the eluate, after it passed through the UV monitor, was flown through the SAXS flow cell. The flow cell consists of a 1.0 mm ID quartz capillary with ~20 μm walls. A coflowing buffer sheath is used to separate sample from the capillary walls, helping prevent radiation damage62 (link). Scattering intensity was recorded using an Eiger2 XE 9 M (Dectris) detector which was placed 3.688 m from the sample giving us access to a q-range of 0.027 Å−1 to 0.42 Å−1. 0.5 s exposures were acquired every 1 s during elution and data were reduced using BioXTAS RAW 2.1.463 (link). Buffer blanks were created by averaging regions flanking the elution peak and subtracted from exposures selected from the elution peak to create the I(q) vs q curves used for subsequent analyses. 3D electron density reconstruction was done using DENSS64 (link) incorporated in RAW 2.1.4.
7
SARS-CoV-2 Spike Glycoprotein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full length SARS-CoV-2 Wuhan-Hu-1 spike glycoprotein (M1-Q1208, GenBank MN90894 ) and RBD constructs (amino acid residues R319-K529, GenBank MN975262.1 ), both with an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag at C-terminus were obtained from Barney S. Graham (NIH Vaccine Research Center) and Aaron G. Schmidt (Ragon Institute), respectively. These mammalian expression vectors were used to transfect Expi293F suspension cells (Thermo Fisher) using polyethylenimine (Polysciences). Transfected cells were allowed to grow in 37°C, 8% CO2 for an additional 5 days before harvesting for purification. Protein was purified in a PBS buffer (pH 7.4) from filtered supernatants by using either StrepTactin resin (IBA) or Cobalt-TALON resin (Takara). Affinity tags were cleaved off from eluted protein samples by HRV 3C protease, and tag removed proteins were further purified by size-exclusion chromatography using a Superose 6 10/300 column (Cytiva) for full length Spike and a Superdex 75 10/300 Increase column (Cytiva) for RBD domain in a PBS buffer (pH 7.4).
8
Characterizing SARS-CoV-2 Main Protease Oligomeric State
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mpro was prepared in 20 mM Tris (pH 7.8) buffer supplemented with 150 mM NaCl and 1 mM EDTA, and for the indicated sample, 1 mM TCEP. The peroxide sample was produced by incubating protein with 1 mM hydrogen peroxide for 5 h prior to injection. Subsequently, protein solutions were spun down at 16,000 g for 5 min and applied to a Cytiva Superdex 75 10/300 increase column using a ÄKTA Pure system from Cytiva. Two peaks are observed in the resulting chromatograms at elution volumes consistent with dimer and monomer species. Concentrations were estimated by peak height at the position of peak maxima, and these were used to fit a two-state dimer dissociation model by least-squares regression. Raw data and processing scripts available upon request.
9
Production of SARS-CoV-2 Spike Proteins
10
Fab Purification and Typhoid Toxin Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified MAbs were concentrated to 20 mg/mL in 0.1 mL of a buffer containing 20 mM sodium phosphate, pH 7.0, and 10 mM EDTA, followed by enzyme digestion using immobilized Papain (Thermo Fisher Scientific) overnight at 37°C. Digested MAbs samples were eluted with 10 mM Tris-HCl, pH 7.5, and the Fabs were differentially purified with Protein G resins away from Fc and undigested mAb. Further purification of Fab was carried out using Superdex 75 10/300 Increase column (Cytiva) using a constant flow of 0.5 mL/min of a buffer containing 10 mM Tris-HCl, pH 7.5. Purified Fab was incubated together with purified typhoid toxin (PltB, PltAE133A, CdtBH160Q) overnight at 4°C and submitted to Superdex 200 10/300 increase column (Cytiva) using a constant flow of 0.5 mL/min of a buffer containing 15 mM Tris-HCl, pH 7.5 and 150 mM NaCl. Fractions corresponding to the Fab-toxin complex were collected and used immediately for Cryo-EM grid/sample preparations for TyTx1 and TyTx4.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!