One-hundred μl of an in vitro transcription (IVT) reaction was set up without or with L7Ae (∼17 μM final). After incubation at 37°C for 10 h, 1 μl of DNase I (10 U/μl) was added to the IVT and the reaction was incubated at 37°C for 30 min. The IVT reaction was then centrifuged (22,000 × g, 20 min, 22°C) and the supernatant was loaded on to a Superdex 200 increase 10/30 GL (GE Healthcare) column using a 100-μl sample loop. The column was run at a flow rate of 0.5 ml/min and with 50 mM HEPES–KOH (pH 7.5), 50 mM MgCl2, 200 mM NaCl, 500 mM KCl and 50 mM potassium acetate (KOAc) as the running buffer (41 (link)).
Superdex 200 increase 10 30 gl
Manufactured by GE Healthcare
The Superdex 200 Increase 10/30 GL is a pre-packed size exclusion chromatography column for laboratory use. It is designed for the separation and purification of proteins, peptides, and other biomolecules based on their molecular size and shape. The column has a bed volume of 24 ml and can be used with a wide range of sample volumes and flow rates.
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Lab products found in correlation
3 protocols using superdex 200 increase 10 30 gl
1
Purification of L7Ae-Bound RNA Complexes
2
Gel Filtration Analysis of LepI Variants
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Proteins used in the experiments, including LepI, LepI-T3A/L10A/Q13A/L14A, LepI-Δ15, and LepI-Δ37-GST, were purified as described above. Pure LepIs were all adjusted to a final concentration of 1 mg/mL before injection into an analytical gelfiltration column (Superdex 200 Increase 10/30 GL; GE Healthcare) equilibrated with buffer containing 20 mm Na2HPO4 and 50 mm NaCl (pH 8.0). The migration positions of the LepI variants were compared with the position of native-LepI. The standard shift was monitored by injecting the following known proteins: thyroglobulin (bovine) (670 kD), γ-globulin (bovine) (158 kD), ovalbumin (chicken) (44 kD), myoglobin (horse) (17 kD), and vitamin B12 (1.35 kD).
3
Molecular Weight Determination of Bacterial Toxin-Antitoxin
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Purified samples of AtYoeB, AtYefM, and co-expressed AtYeoB-YefM were analyzed for absolute molecular weight determination using a Wyatt miniDAWN Treos system integrated with size exclusion chromatography and a refractive index detector, all maintained at room temperature. The sizing column, a Superdex 200 Increase 10/30 GL (GE Healthcare), was equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl buffer. The resulting light scattering profiles were analyzed using the ASTRA software (v 6.1, Wyatt Technologies) following manufacturer’s recommendations; concentrations were determined based on the signal from the refractive index detector, as AtYefM contains no tryptophan amino acids, making concentration calculations from absorbance at 280 nm error prone. The resulting data were ported to GraphPad Prism (v 6.0d) and replotted for graphic presentation.
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