The largest database of trusted experimental protocols

14 protocols using superdex 200 10 300

1

Analytical SEC and Thermal Stability of Vpb4Da2

Check if the same lab product or an alternative is used in the 5 most similar protocols
Analytical size-exclusion chromatography (SEC) of the full-length and trypsin-processed Vpb4Da2 was performed on an AKTA pure™ using a Superdex-200 10/300 GL increase (Cytiva) equilibrated at 4°C with 25 mM Na carbonate pH 9.5 and 100 mM NaCl. Protein samples were injected at the flow rate of 0.5 mL/min. Superdex-200 10/300 was calibrated using Ferritin (440, 000 Da), Aldolase (158, 000 Da), Conalbumin (75,000 Da), Ovalbumin (44, 000 Da), and Blue dextran 2000, which were all from Cytiva. Kav = (Ve-Vo)/Vc-Vo), with Ve = elution volume, Vo = void volume (8.07 mL), and Vc = Column volume (24 mL) were used. Calibration curve equation, Y = -03896x + 1.2061 (R2 = 0.9225) was used to calculate the molecular weight for full length and trypsin processed Vpb4Da2.
Thermal stability of the purified wild-type Vpb4Da2 and variants in protein storage buffer was performed in triplicate reactions using 25 μL of 0.5 mg/mL protein and 5 μL of 10x SYPRO™ Orange (Thermo Scientific). The CFX96™ real-time PCR detection system (Bio-Rad) was used to follow a thermal denaturation gradient of 20°C to 95°C with an increment of 0.5°C per 5 Sec. Fluorescence data were fit using the CFX manager 2.1 (Bio-Rad).
+ Open protocol
+ Expand
2

Purification of Recombinant Parkin Oligomers

Check if the same lab product or an alternative is used in the 5 most similar protocols
The synthetic gene prkn2_rat (GeneArt) was cloned into pMK (kanR) using SacI and KpnI cloning sites. The full-length gene was cleaved by BamHI and XhoI restriction sites and ligated into pGEX-6P1 vector. The recombinant proteins of construct were overexpressed in E. coli KRX cells and grown at 37 °C in lysogeny broth medium containing 100 mg/l ampicillin until the A600 level of 0.6 was recorded. Proteins expressions were induced by addition of 0.7 mM IPTG, supplemented with 0.5 mM ZnCl2. Following induction, cells were grown overnight at 16 °C. The cells were harvested and lysed by sonication on ice in buffer A (20 mM Tris-HCl (pH 7.4), 120 mM NaCl) supplemented with 1% Triton X-100 and 2 mM PMSF.
The cell lysates were centrifuged at 48,000 g for 30 min, at 4 °C. The supernatant of GST-parkin was purified on a 20 ml GST column and eluted with 10 mM reduced glutathione. The pull-down fractions were concentrated using Amicon Ultra concentrators with 10 kDa Millipore membrane and separated by gel-filtration on Superdex200 10/300 (Cytiva, Uppsala, Sweden). Purified on affinity chromatography, parkin was eluted from size-exclusion Superdex200 column in V0 as oligomers with molecular weight not less than 1000 kDa.
+ Open protocol
+ Expand
3

Purification of BTB/POZ Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
KCTD1 FL, BTB/POZ1, BTB/POZ11 and BTB/POZ15 proteins were produced as described elsewhere [12 (link),23 (link),25 (link),45 (link)].
The BTB/POZ domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states [45] (link).
The full-length sequence of KCASH2 was cloned in pGEX4T3 using the restriction sites BamHI and EcoRI. The recombinant protein GST-KCASH2 was purified by a GST-Trap FF (Cytiva, Milan, Italy). The fraction of interest was dialyzed against 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM DTT at 4°C and successively digested with the specific protease (Thrombin, SIGMA Aldrich). Successively, the sample was loaded again on the GST-affinity chromatography and finally obtained pure on a gel-filtration chromatography (Superdex 200 10 × 300, Cytiva, Milan, Italy).
+ Open protocol
+ Expand
4

Recombinant SARS-CoV-2 Protein Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
Plasmids coding for recombinant proteins were transiently transfected in Expi293F™ cells (Thermo Fischer) using FectoPRO® DNA transfection reagent (Polyplus), according to the manufacturer’s instructions. Cells were incubated at 37°C for 5 days and then the culture was centrifuged and the supernatant was concentrated. Proteins were purified from the supernatant by affinity chromatography using StrepTactin columns (IBA) (SARS-CoV-2 S) or His-Trap™ Excel columns (Cytiva) (SARS-CoV-2 RBD). A final step of size-exclusion chromatography (SEC) in PBS was also performed, using either a Superose6 10/300 column (Cytiva) for the SARS-CoV-2 S, or a Superdex200 10/300 (Cytiva) for the SARS-CoV-2 RBD.
+ Open protocol
+ Expand
5

Purification of LmCpfC Protein Complexes

Check if the same lab product or an alternative is used in the 5 most similar protocols

LmCpfC wild‐type and variants were generated, expressed, and purified as reported previously.17 Prior to measurements or drop setting for crystallization the reconstituted cpIII‐LmCpfC complex was purified to monodispersity using a size exclusion chromatography column (Superdex 200 16/600, Cytiva). All samples were additionally checked for purity and monomeric state using an HPLC (Shimadzu prominence LC20)‐SEC (Superdex 200 10/300, Cytiva)‐MALS (Wyatt, Heleos Dawn8+) system.
+ Open protocol
+ Expand
6

Protein Characterization by SEC-MALS

Check if the same lab product or an alternative is used in the 5 most similar protocols
An Agilent 1260 Infinity II HPLC system was used to inject 50 μl of protein (ranging from 0.5 to 2.5 mg/ml) onto a Superdex 200 10/300 gel filtration column (Cytiva) coupled to a Wyatt miniDawn multi-angle light scattering system and Wyatt Optilab differential refractive index (dRI) detector. A dn/dc value of 0.185 was used, UV extinction coefficients were calculated from RI and molecular weights were calculated using Zimm plot extrapolation. Astra v8.1 was used for data analysis. The following buffer was used in all experiments: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM TCEP.
+ Open protocol
+ Expand
7

Labeling Recombinant Histones for Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols
Human recombinant histones were purchased from “The Histone Source” which operates at Colorado State University (https://histonesource-colostate.nbsstore.net/). Octamer reconstitution was performed with WT histones with the exception of H3(C110A) and H2A(K119C) which is required for labeling with Cy5 maleimide. The histone octamer was refolded by adding each of the histones together with H2A/H2B in 10% excess of H3/H4 as previously described.63 (link) The histone octamer was labeled with Cy5-maleimide (GE Healthcare) and purified with a Superdex 200 10/300 (Cytiva) size exclusion column as previously described.64 (link)
+ Open protocol
+ Expand
8

Recombinant SARS-CoV-2 RBD Protein Production

Check if the same lab product or an alternative is used in the 5 most similar protocols
The plasmids coding for recombinant proteins were transiently transfected in Expi293F™ cells (Thermo Fischer) using FectoPRO® DNA transfection reagent (Polyplus), according to the manufacturer’s instructions. The cells were incubated at 37 °C for 5 days and then the culture was centrifuged and the supernatant was concentrated. The proteins were purified from the supernatant by affinity chromatography using His-Trap™ Excel columns (Cytiva) (SARS-CoV-2 RBD). A final step of size-exclusion chromatography (SEC) in PBS was also performed, using a Superdex200 10/300 (Cytiva) for the SARS-CoV-2 RBD.
+ Open protocol
+ Expand
9

Molecular Weight Determination of Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
To determine the molecular weight of the PPP2, AGDC1, and AGDC1 E88A, samples of each protein were analyzed by size exclusion chromatography using a Superdex 200 10/300 (Cytiva, Marlborough, MA, USA) column mounted on a Bio-rad (Hercules, CA, USA) NGC chromatography system. SEC runs of the proteins were done in the presence (25 mM Tris 7.5, 50 mM KCl, 100 mM NaCl) or absence (25 mM Tris 7.5, 100 mM NaCl) of potassium. Bio-rad Gel Filtration Standard (catalog number 1511901) was used as a standard. All proteins eluted as a single peak. Chromatograms were analyzed and plotted using a home-written Python script.
+ Open protocol
+ Expand
10

Purification of His-Tagged Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
All constructs were expressed in BL21-Rosetta(DE3) (Novagen). Transformed cells were grown to an absorbance of 0.7 at 600 nm, induced with 0.8 mM IPTG, and expressed overnight at 18 °C. Cells were resuspended in buffer containing 20 mM Tris (pH 8.0), 500 mM NaCl, 5 mM beta-mercaptoethanol, and 20 mM imidazole and lysed with sonication. Lysate was clarified with centrifugation, and supernatant was subjected to nickel affinity chromatography to isolate the His-tagged proteins. Proteins were eluted with 200 mM imidazole. Proteins were then subjected to ion-exchange chromatography using a HiTrap Q column (Cytiva). After ion exchange, proteins were concentrated using a spin concentrator (Millipore). Concentrated proteins were either loaded directly onto a Superdex 200 10/300 (Cytiva) in buffer with 20 mM Hepes (pH 7.5), 150 mM NaCl, and 1 mM Tris(2-carboxyethyl)phosphine (TCEP), or incubated with lambda phosphatase (New England BioLabs) for 1 h at room temperature or 16 h at 4 °C to dephosphorylate prior to loading onto a Superdex 200 10/300 in buffer with 20 mM Hepes (pH 7.5), 150 mM NaCl, and 1 mM TCEP.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!