His-ICDH recombinant proteins (~10 μg) were fractionated using Superose 12 (GE Healthcare, Buc, France) column equilibrated in Protein Buffer (50 mM Tris pH 7.5, 5 mM MgCl2) containing 150 mM NaCl. The protein standards were aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa) (GE Healthcare).
Superose 12
Manufactured by GE Healthcare
Sourced in Sweden
Superose 12 is a gel filtration chromatography column used for the separation and purification of biomolecules based on their molecular size. It is composed of highly cross-linked agarose beads that provide a porous matrix for the separation process. Superose 12 is designed to efficiently separate molecules within a molecular weight range of 1,000 to 300,000 Daltons.
Automatically generated - may contain errors
Lab products found in correlation
Superose 6, by GE Healthcare (3 mentions)
Aldolase, by GE Healthcare (1 mentions)
Conalbumin, by GE Healthcare (1 mentions)
Ovalbumin (ova), by GE Healthcare (1 mentions)
Hitrap q, by GE Healthcare (1 mentions)
Superose 12 10 30, by GE Healthcare (1 mentions)
Ni nta column, by Qiagen (1 mentions)
Akta prime fplc system, by GE Healthcare (1 mentions)
Gel filtration calibration kits lmw and hmw, by GE Healthcare (1 mentions)
Resource s, by GE Healthcare (1 mentions)
Lysozyme, by Euromedex (1 mentions)
Dnase 1 grade 2, by Roche (1 mentions)
Hitrap chelating, by GE Healthcare (1 mentions)
Rnase, by Roche (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Sephacryl 100, by GE Healthcare (1 mentions)
Mono q, by GE Healthcare (1 mentions)
Kta purifier fplc, by GE Healthcare (1 mentions)
Source s, by GE Healthcare (1 mentions)
Antibody against pml, by Abcam (1 mentions)
15 protocols using superose 12
1
Protein Fractionation using Superose 12
2
Purification and Oligomerization of α-Synuclein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human WT α-Syn with N-terminal his-tag in pET28a vector was expressed and purified from BL21-STAR E. coli using a Ni-NTA column (Qiagen) followed by an anion-exchange chromatography (HiTrapQ, GE Healthcare). Pure α-Syn monomers were obtained by SEC (Superose 12 10/30, GE Healthcare) immediately prior to use. The purity of α-Syn monomers (>98%) was determined by SDS-PAGE and quantified by densitometry (ImageJ, NIH). Crude α-Syn aggregates/oligomers were generated by incubating monomeric α-Syn (3 mg/mL) in PBS (20 mM NaPO4 pH 7.4, 140 mM NaCl) at 37 °C for 7 h. After oligomerization, the oligomers were separated from the remaining monomers by SEC (Superose 12, GE Healthcare) and verified A11+ by dot blot.
3
Protein Complex Formation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of purified recombinant proteins were applied to Superose-12 (GE Healthcare) gel filtration column and analyzed using an AKTA Prime FPLC system (GE Healthcare). For analysis of RecA–AlkB complex, 0.5 mg (35 μM) of AlkB was mixed with 0.735 mg (35 μM) of RecA in 0.5 ml buffer containing 25 mM NaCl and 20 mM HEPES, pH 7.0 or 20 mM Tris–HCl, pH 8.0 or 9.0. E. coli RecA (EcRecA) was purchased from New England Biolabs (M0249L). For the analysis of EcRecA, 0.5 mg (35 μM) of AlkB was mixed with 0.735 mg (35 μM) of RecA in 0.5 ml buffer containing 100 mM NaCl and 20 mM HEPES, pH 7.0 or 20 mM Tris–HCl, pH 8.0. For the SEC analysis of RecA titration 20 μM of AlkB was mixed with, 20, 40, 80, 160 μM RecA protein. For AlkB titration 20 μM of RecA was mixed with, 20, 40, 80 μM AlkB protein. The samples were analyzed with flow rate of 0.3 ml/min and 0.5 ml fractions were collected.
4
Molecular Mass Evaluation of Recombinant Shewasin Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The molecular masses of purified active recombinant shewasin D and shewasin D_D37A were evaluated by size-exclusion chromatography under non-denaturing conditions on a Superose 12 (GE Healthcare Life Sciences) column equilibrated with 20 mM Tris-HCl buffer pH 8.0 with 150 mM NaCl in a DuoFlow-BioRad FPLC system. The column was calibrated with Gel Filtration HMW and LMW calibration kits (GE Healthcare Life Sciences), using as molecular mass markers aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa).
5
Recombinant Expression and Purification of P. abyssi Q9UZY3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four recombinant versions of P. abyssi Q9UZY3 were synthesized and cloned (Genecust) as described in Figure S2 . Following the same protocol as for PAN expression, proteins were overexpressed in E. coli BL21(DE3) and cell pellets were resuspended into Buffer 2 (20 mM Tris–HCl, pH 8.0, 150 mM NaCl), complemented with 0.1% Triton X‐100, 25 mM MgSO4, 0.25 mg ml−1 lysozyme (Euromedex), 0.05 mg ml−1 DNase I grade II (Roche), 0.2 mg ml−1 RNase (Roche) and EDTA‐free protease inhibitor (cOmplete™, Roche). The cells were disrupted by sonication and incubated at 25°C for 30 min. Then the cell extract was treated by heating at 70°C for 15 min and the lysate was clarified by centrifugation at 10,000×g for 1 h. His‐tagged Q9UZY3 was purified by using (i) an affinity column (5‐ml HiTrap Chelating, GE Healthcare) with a linear gradient of 20–250 mM imidazole; (ii) a cation exchange column (Resource S, GE Healthcare) with a linear gradient of 50–500 mM NaCl and (iii) a size exclusion column (Superose 12, GE Healthcare) with elution in Buffer 2. For the untagged Q9UZY3, only the purification steps (ii) and (iii) were performed. The final Q9UZY3 concentration was calculated by measuring the absorbance at 280 nm using the predicted extinction coefficient (ProtParam, ExPASy) listed in Table S1 . The primers and cloning strategies for expression in bacteria cells are listed in Table S2 .
6
Molar Mass Distribution Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The molar mass distribution was evaluated by SEC with a system comprised of three 10 × 300 mm columns in series (Superose 6 → Superdex 200 → Superose 12, GE Healthcare Life Sciences, Uppsala, Sweden). The eluent was 0.5 M NaCl + 0.01 NaN3 at 0.6 mL∙min−1. A differential refractive index detector was used as the mass detector for the molar mass evaluation; absorbance at 310 nm was used to qualitatively evaluate the presence of ferulic acid and its derivatives. CPCWin 32 (a.h. group, Graz, Austria) was used to perform the calculations on the raw chromatographic data. The molar masses are reported as dextran equivalent.
7
Analytical Size Exclusion Chromatography
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
0.1 mL of sample (0.5 mg/mL of proteins) was loaded onto Superose 12 (GE Healthcare) analytical and preparative column (30 cm×1 cm) equilibrated by 25 mM Tris-HCl (pH 7.4). The flow rate after sample application was 0.5 mL/min. Marker proteins were albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa).
8
Expression and Purification of Odorant-Binding Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For expression of recombinant proteins, each pET-5b vector containing the appropriate odorant-binding protein (OBP) sequence was used to transform BL21(DE3)pLysS and BL21(DE3)Rosetta-gami E. coli cells, for OBP18 and OBP16 respectively. Protein expression was induced by addition of IPTG to a final concentration of 0.4 mM when the culture had reached a value of O.D.600 = 0.8. Cells were grown for further 2 h at 37°C, in the case of OBP18, while they were grown overnight at 30°C for OBP16 expression. They were then harvested by centrifugation and sonicated. After centrifugation, OBP16 was soluble while OBP18 was present as inclusion bodies. To solubilize it, the pellet from 1 L of culture was dissolved in 10 mL of 8 M urea, 1 mM DTT in 50 mM Tris buffer, pH 7.4, then diluted to 100 mL with Tris buffer and dialysed three times against Tris buffer.
Purification of the proteins was accomplished by combinations of chromatographic steps anion-exchange resins, such as DE-52 (Whatman), QFF or Mono-Q (GE-Healthcare), followed by gel filtration on Sephacryl-100 or Superose-12 (GE-Healthcare) along with standard protocols previously adopted for other odorant-binding proteins [63 (link),64 (link)]. The electrophoretic analysis of crude bacterial pellets and representative fractions from the last purification steps for OBP16 and OBP18 are shown in Additional file6 : Figure S1.
Purification of the proteins was accomplished by combinations of chromatographic steps anion-exchange resins, such as DE-52 (Whatman), QFF or Mono-Q (GE-Healthcare), followed by gel filtration on Sephacryl-100 or Superose-12 (GE-Healthcare) along with standard protocols previously adopted for other odorant-binding proteins [63 (link),64 (link)]. The electrophoretic analysis of crude bacterial pellets and representative fractions from the last purification steps for OBP16 and OBP18 are shown in Additional file
9
Purification and Oligomeric Analysis of HydF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of C.a. HydF
were analyzed via Superose 12 (GE Healthcare) size-exclusion chromatography
(HR 10/30 column; 1 cm inside diameter, 30 cm length) at room temperature
within a Coy anaerobic chamber, maintained as described above. Column
equilibration into a 50 mM HEPES (pH 7.4), 0.3 M KCl, 5% glycerol
buffer was accomplished using an ÄKTA Purifier FPLC instrument
(GE Healthcare) at a flow rate of 0.2 mL/min. Sample runs were performed
at least in duplicate on one of two columns, with slightly different
bed volumes. The sample oligomeric content was calibrated against
a Bio-Rad standard (#151-1901) that contained thyroglobin (bovine),
γ-globulin (bovine), ovalbumin (chicken), myoglobin (horse),
and vitamin B12. Samples were injected either into the
mixer port of the FPLC with a ∼2 ft tube (0.076 cm inside diameter)
lead on the column or directly onto the column. Under these conditions,
tetrameric (∼189 kDa) and dimeric (∼94.5 kDa) HydF species
eluted with retention volumes of ∼9–11 and ∼10–12
mL, respectively, with variability due to altered injection techniques
or the specific column used. Separate calibration curves using the
Bio-Rad standard solution were created for each sample injection to
ensure accurate assessment of HydF oligomeric forms.
were analyzed via Superose 12 (GE Healthcare) size-exclusion chromatography
(HR 10/30 column; 1 cm inside diameter, 30 cm length) at room temperature
within a Coy anaerobic chamber, maintained as described above. Column
equilibration into a 50 mM HEPES (pH 7.4), 0.3 M KCl, 5% glycerol
buffer was accomplished using an ÄKTA Purifier FPLC instrument
(GE Healthcare) at a flow rate of 0.2 mL/min. Sample runs were performed
at least in duplicate on one of two columns, with slightly different
bed volumes. The sample oligomeric content was calibrated against
a Bio-Rad standard (#151-1901) that contained thyroglobin (bovine),
γ-globulin (bovine), ovalbumin (chicken), myoglobin (horse),
and vitamin B12. Samples were injected either into the
mixer port of the FPLC with a ∼2 ft tube (0.076 cm inside diameter)
lead on the column or directly onto the column. Under these conditions,
tetrameric (∼189 kDa) and dimeric (∼94.5 kDa) HydF species
eluted with retention volumes of ∼9–11 and ∼10–12
mL, respectively, with variability due to altered injection techniques
or the specific column used. Separate calibration curves using the
Bio-Rad standard solution were created for each sample injection to
ensure accurate assessment of HydF oligomeric forms.
10
Gel Filtration of HydF Protein
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!