DnaJB8F→S, JD1–82, and CTD170–232 constructs at a concentration of 5.1, 5.0, and 6.6 mg/mL, respectively, in 1× PBS were filtered through a 0.1-μm filter to remove larger impurities. Each sample was further filtered using a 0.22-μm centrifugal filter before 100 μL was applied to a Superdex 200 Increase 10/300 column equilibrated in 1× PBS with 1 mM TCEP. The column was in line with a Shimadzu UV detector, a Wyatt TREOS II light-scattering detector, and a Wyatt Optilab tREX differential-refractive-index detector. The flow rate was 0.5 mL/min. The data were analyzed with Wyatt’s ASTRA software version 7.1.0.29. SEDFIT65 (link) was used to calculate the dn/dc of the protein.
T rex differential refractive index detector
Manufactured by Wyatt Technology
Sourced in United States
The T-rEx differential refractive index detector is a laboratory instrument used to measure changes in the refractive index of a sample. It operates by comparing the refractive index of the sample to a reference solution, providing a sensitive and accurate measurement of refractive index differences.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 75 increase 10 300 gl column, by GE Healthcare (2 mentions)
Astra software version 5, by Wyatt Technology (2 mentions)
Qels dynamic light scattering detector, by Wyatt Technology (2 mentions)
Dawn heleos 2 multi angle static light scattering detector, by Wyatt Technology (2 mentions)
Uv detector, by Wyatt Technology (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
Mwco 10 kda, by Merck Group (1 mentions)
Mwco concentrator, by Merck Group (1 mentions)
Tris hcl ph 8, by Merck Group (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Wyattqels, by Wyatt Technology (1 mentions)
4 protocols using t rex differential refractive index detector
1
Size-Exclusion Chromatography of Protein Constructs
2
Oligomeric State Determination of YebY
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SEC–MALS was used to determine the oligomeric state of untagged YebY. Aliquots of YebY were thawed and buffer exchanged using 10 kDa MWCO (molecular weight cutoff) concentrators (Millipore) into 20 mM Hepes, pH 7.0, 100 mM NaCl. Samples (250 μl, ∼300 μM) were prepared at room temperature immediately before use and analyzed using an Agilent 1260 series HPLC system equipped with diode array detection absorbance in-line with a DAWN HELEOS II multiangle static light scattering detector (Wyatt Technology), a QELS dynamic light scattering detector (Wyatt Technology), and a T-rEx differential refractive index detector (Wyatt Technology). The samples were injected onto a Superdex 75 Increase 10/300 GL column (GE Healthcare) that had been pre-equilibrated in running buffer (20 mM Hepes, pH 7.0, 100 mM NaCl). The buffer was stored at room temperature, and the column was kept at 8 °C. Each sample was run at 0.4 ml min−1 for 60 min. Data processing and analysis were performed using Astra software, version 5.3.4 (Wyatt Technology).
3
SEC-MALS Analysis of PmoD Oligomers
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SEC-MALS was used to determine the molar masses of the two PmoD species resolved by SEC. Each peak was pooled and concentrated to 2–4 mg mL−1 in 10 kDa MWCO concentrators (Millipore). Samples were analyzed using an Agilent 1260 series high-performance liquid chromatography (HPLC) system equipped with diode-array detection absorbance in-line with a DAWN HELEOS II multi-angle static light scattering detector (Wyatt Technology), a QELS dynamic light scattering detector (Wyatt Technology), and a T-rEx differential refractive index detector (Wyatt Technology). Then, 300 μL protein was applied to a Superdex 75 Increase 10/300 GL column (GE Healthcare) that had been pre-equilibrated in a mobile phase consisting of 20 mM Tris, pH 7.0. The mobile phase and protein sample prior to injection were maintained at 4 °C and the column was kept at 8 °C. Each sample was run at 0.4 mL min−1 for 60 min. Optical spectra were also recorded at the apex of each peak. Data were processed and analyzed using Astra software version 5.3.4 (Wyatt Technology).
4
SEC-MALS Analysis of GlnH-His6 Protein
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SEC-MALS analysis involved an injection of 50 µL of the 100 µM GlnH-His6 solution in buffer E onto a Superdex 200 5/150 HiLoad size-exclusion column (GE Healthcare, Chicago, IL, USA) pre-equilibrated with buffer E flowing at 0.15 mL/min. The eluate was passed through a Shimadzu HPLC in-line DAWN HELEOS light scattering detector, an Optilab T-rEX differential refractive index detector and a quasi-elastic light scattering detector (WyattQELS, Wyatt Technology Corporation, Santa Barbara, CA, USA). The MALS 8.5 and 30% w/v polyethylene glycol (PEG) 4000. After two days, needle-like crystals also appeared in condition H3 of the JCSG+ Suite screen, containing 0.17 M ammonium sulfate, 25.5% w/v PEG 4,000 and 15% v/v glycerol. Manual optimization resulted in larger, rod-like crystals in a condition containing 0.2 M sodium acetate (Merck, Darmstadt, Germany), 0.05 M Tris-HCl pH 8.5 and 28% w/v PEG 4,000 (Sigma-Aldrich, MO, USA).
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