α-Syn aggregates showing spherical shape were generated as previously described [33 (link)]. Purified 10 µM α-syn was incubated with 200 µM dopamine in 20 mM sodium phosphate buffer (pH 7) for 3 days at 37°C with constant agitation. The sample was centrifuged at 14,000 g for 10 min at 4°C to remove insoluble aggregates. The supernatant was concentrated and loaded on a Superdex 200 10/30 GL column (GE Healthcare, UK) to separate aggregates from monomer using PBS in NGCTM Chromatography Systems (Bio-Rad, USA). Fractions containing aggregates were concentrated again and stored at 4°C until use. Another spherical form of aggregates was prepared as described previously [38 (link)]. Briefly, α-syn (12 mg/ml) was incubated in PBS at 37°C for 24 h without agitation. The excess of monomeric protein and the low levels of oligomer were separated by size-exclusion chromatography. α-Syn preformed fibrils (PFF) were prepared by agitating α-syn (5 mg/ml) in PBS at 37°C. After 7 days of incubation, α-syn aggregates were sonicated for 30 s and the monomer and PFF were separated by size-exclusion chromatography. The fractions containing monomer and PFF were kept at -80°C. To generate Aβ aggregates, Aβ1–42 peptide (GenScript, USA) was dissolved in HPLC grade water at 1 mg/mL and 10 µM peptide was incubated in PBS at 37°C for 5 days.
Superdex 200 10 30 gl column
Manufactured by GE Healthcare
Sourced in United States
The Superdex 200 10/30 GL column is a size exclusion chromatography column used for the separation and purification of proteins, peptides, and other biomolecules. The column has a separation range of 10,000 to 600,000 daltons and a bed volume of 24 ml. It is compatible with a variety of aqueous buffer systems and can be used with standard FPLC or HPLC systems.
Automatically generated - may contain errors
Lab products found in correlation
Kta purifier, by GE Healthcare (2 mentions)
Nanobrook 90plus particle size analyzer, by Brookhaven Instruments (2 mentions)
Bic particle sizing software, by Brookhaven Instruments (2 mentions)
Astra 6, by Wyatt Technology (2 mentions)
Aβ1 42 peptide, by GenScript (1 mentions)
Ni2 affinity chromatography, by GE Healthcare (1 mentions)
Modulustm 2 microplate reader, by Promega (1 mentions)
High molecular weight gel filtration calibration kit, by GE Healthcare (1 mentions)
Kta pure fplc system, by GE Healthcare (1 mentions)
Carbonic anhydrase, by Merck Group (1 mentions)
Beta amylase, by Merck Group (1 mentions)
Micro bio spin size exclusion columns, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Shodex ri 101, by Resonac (1 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
Minidawn treos, by Wyatt Technology (1 mentions)
Glutathione beads, by GE Healthcare (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
K2 summit direct detector, by Ametek (1 mentions)
18 protocols using superdex 200 10 30 gl column
1
Preparation of Protein Aggregates
2
Purification of Truncated PARN Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmid containing the cDNA sequence of the wild type human PARN was kindly provided by Professor Anders Virtanen (Uppsala University, Sweden). The truncated proteins p60 (residues 1–520 AA) and p46 (residues 1–446 AA) were constructed by standard protocols of mutagenesis using the following primers: p60-forward, 5′-CGATGTCACATATGGAGATAATCAGGAGC-3'; p60-reverse 5′-GATCCTCGAGCTACTTCTCTTCCTGTTTTC-3'; p46-forward, 5′-GCTACTCGAGCTTCTCTTCCTGTTTTC-3'; and p46-reverse, 5′-GATCGTCGACTTAATGATCACGTTTAGGCTGC-3'. The obtained genes were cloned to the expression vector pET-28a (Novagen) and verified by sequencing. The recombinant proteins were overexpressed in Escherichia coli BL21 (DE3) (Stratagene, Heidelberg, Germany) and purified as described previously [18 (link),28 (link)]. In brief, the expression of the proteins was induced by 0.1 mM IPTG at 16 °C for 24 h. The proteins were isolated from the supernatant fraction of cell lysates by Ni2+-affinity chromatography (GE Healthcare),. The final products were purified using a Superdex 200 10/30 GL column equipped on an ÄKTA purifier (GE Healthcare). The protein concentration was determined according to the Bradford method [29 (link)]. The protein solutions used for analysis were prepared in buffer A containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM DTT, 0.2 mM EDTA and 20% (v/v) glycerol.
3
Characterization of FGCaMP7 Oligomeric State
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were expressed, purified on a low-scale format, and characterized as described in references [8 (link),19 (link)].
To determine the oligomeric state of the FGCaMP7, the protein (1.7 mg/mL concentration) was applied to a Superdex 200 10/30 GL column (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mM Tris-HCl, pH 7.5, 1 mM CaCl2, or 2 mM EDTA.
To determine the oligomeric state of the FGCaMP7, the protein (1.7 mg/mL concentration) was applied to a Superdex 200 10/30 GL column (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mM Tris-HCl, pH 7.5, 1 mM CaCl2, or 2 mM EDTA.
4
Characterization of NCaMP7 Calcium Sensor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were expressed, purified, and characterized as described in reference [9 (link)].
To assess the equilibrium Kd, the purified proteins (2 µg/mL) were added to buffer A (30 mM HEPES, 100 mM KCl, pH 7.2) supplemented with 10 mM EGTA (zero free Ca2+) pre-mixed in various ratios with buffer A supplemented with 10 mM Ca-EGTA (39 µM free Ca2+). After 20 min of equilibration at r.t. their green fluorescence was measured on ModulusTM II Microplate Reader (TurnerBiosystems, Sunnyvale, CA, USA). The titration of indicators with Ca2+ ions in the presence of 1 mM Mg2+ was done similarly, except buffer A supplemented with 1 mM MgCl2 and 10 mM EGTA (zero free Ca2+) was mixed with buffer A supplemented with 1 mM MgCl2, 10 mM Ca-EGTA (39 µM free Ca2+).
To determine the oligomeric state of the NCaMP7, the protein (1.7 mg/mL concentration) was applied to a Superdex 200 10/30 GL column (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mM Tris-HCl, pH 7.5, 5 mM CaCl2.
To assess the equilibrium Kd, the purified proteins (2 µg/mL) were added to buffer A (30 mM HEPES, 100 mM KCl, pH 7.2) supplemented with 10 mM EGTA (zero free Ca2+) pre-mixed in various ratios with buffer A supplemented with 10 mM Ca-EGTA (39 µM free Ca2+). After 20 min of equilibration at r.t. their green fluorescence was measured on ModulusTM II Microplate Reader (TurnerBiosystems, Sunnyvale, CA, USA). The titration of indicators with Ca2+ ions in the presence of 1 mM Mg2+ was done similarly, except buffer A supplemented with 1 mM MgCl2 and 10 mM EGTA (zero free Ca2+) was mixed with buffer A supplemented with 1 mM MgCl2, 10 mM Ca-EGTA (39 µM free Ca2+).
To determine the oligomeric state of the NCaMP7, the protein (1.7 mg/mL concentration) was applied to a Superdex 200 10/30 GL column (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mM Tris-HCl, pH 7.5, 5 mM CaCl2.
5
Purification of Monomeric and Oligomeric α-Synuclein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human recombinant αSN was produced as described previously [40 (link)]. In order to prepare monomer and oligomer, lyophilized αSN was dissolved in PBS (7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 140 mM NaCl, pH 7.4) to a final concentration of 10 mg/ml (690µM) and incubated for 30 min on ice. The dissolved protein was centrifuged on a tabletop centrifuge at 14,800 rpm for 5 min at 4°C and afterwards loaded on a Superdex 200 10/30 GL column (GE Healthcare) and eluted with 0.5 ml/min PBS [41 (link)]. Oligomers were collected from 17–22 min, and monomer αSN was collected at 35 min (Fig. 1 ) Protein concentrations of the fractions were determined by Bicinchoninic acid protein concentration assay (BCA) (Pierce).
6
Oligomeric State of Tg Cystathionine Beta-Synthase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The oligomeric state of TgCBS was investigated via gel filtration on a GE Healthcare Superdex 200 10/30 GL column in 20 mM sodium phosphate buffer pH 8.5, 150 mM NaCl and 0.1 mM DTT. High molecular weight gel filtration calibration kit (GE Healthcare) was used to construct a calibration curve, following protocols in Refs.50 (link),51 (link).
7
Characterizing Heterotrimer Size by SEC and DLS
8
Site-specific Labeling of A11 cMb with Maleimide-Cy5.5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A11 cMb was site-specifically labeled with maleimide-Cy5.5 (mal-Cy5.5) by selective reduction of and conjugation to C-terminal cysteines. In a typical reaction, 200 µg of protein at 1 mg/mL in phosphate-buffered saline (PBS) was reduced using a 2-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP, Pierce) for 30 min at room temperature. Equimolar mal-Cy5.5 (Amersham, GE Healthcare) was added to the reduced A11 cMb for 2 h at room temperature. Excess mal-Cy5.5 was removed using a Micro Bio-Spin™ Size Exclusion Columns (Bio-Rad) pre-equilibrated with PBS. Dye-to-protein ratio (D:P) was determined by measuring the protein (280 nm) and Cy5.5 absorbance (675 nm) with a spectrophotometer (NanoDrop 2000). Successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC).
For SEC, a Superdex 200 10/30 GL column (GE Healthcare) was used with an ÄKTA purifier (GE Healthcare) with PBS as the mobile phase (0.5 mL/min). Absorbance at 280 nm (protein) and 675 nm (Cy5.5) was recorded. The following protein standards were used: beta-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) (Sigma).
For SEC, a Superdex 200 10/30 GL column (GE Healthcare) was used with an ÄKTA purifier (GE Healthcare) with PBS as the mobile phase (0.5 mL/min). Absorbance at 280 nm (protein) and 675 nm (Cy5.5) was recorded. The following protein standards were used: beta-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) (Sigma).
9
Size-Exclusion Chromatography of KaiB-KaiC Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KaiB (0.24 mg/ml) and KaiC (1.2 mg/ml) had been pre-incubated separately at 30 °C for 2 h, and then equal volumes of two samples were mixed together (t = 0). An aliquot (500 μL) of the mixture was loaded onto a Superdex 200 10/30 GL column (GE Healthcare) immediately after the mixing (t = ~2 min), or after further incubation at 30 °C for 18 h. The column was equilibrated in a buffer containing 50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, and 0.5 mM ATP or AMP-PNP at pH 8.0. Analysis was performed at a flow rate of 0.5 mL/min at room temperature.
10
Probing TPX2-Tubulin Interactions via SEC-MALS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
35 µM His6-mCherry-tagged TPX2 α3–α7 and35 µM untagged TPX2 α5–α7 were subjected to SEC using a Superdex 200 10/30 GL column (GE Healthcare) equilibrated with CSF-XB at 4°C. The eluate was directed to a DAWN HELEOS-II MALS instrument in tandem with an OptiLab TrEX differential refractometer for real-time analysis. Molar mass was calculated using ASTRA 6.0 (Wyatt Technology) software package. For testing the interaction between TPX2 and tubulin, 10 µM His6-mCherry-tagged TPX2 α3–α7 and 10 µM His-6-GFP-tagged TPX2 α5–α7 were buffer-exchanged into ice-cold BRB80 with 6 mM BME and then centrifuged at 270,00 g for 15 min at 4°C. Porcine brain tubulin at 0.7 mg/ml in BRB80 with 1 mM GTP was also centrifuged with the above parameters. Both TPX2 proteins and tubulin were diluted twofold in BRB80 with 6 mM BME and individually run on a Superdex 200 10/30 GL column equilibrated with BRB80. Subsequently, both TPX2 proteins were separately mixed in equal volume with the precleared tubulin, incubated on ice for 20 min, and also analyzed via SEC. Absorbance at 280, 488, and 561 nm was simultaneously measured for all samples eluting from the column.
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!