For the determination of the oligomeric state of VapG, the protein was analysed by SEC-MALLS. Samples of protein at concentrations of 4 mg/ml were loaded onto a Superdex S75 10/300 gel-filtration column equilibrated at 0.5 ml min−1 with a mobile phase consisting of 50 mM Tris–HCl pH 8.0, 150 mM NaCl. The eluate was passed through an SPD20A UV–vis detector, a Wyatt Dawn HELEOS-II 18-angle light-scattering detector and a Wyatt Optilab rEX refractive index monitor with the system driven by a Shimadzu HPLC system comprising an LC-20AD pump. The data were processed and molecular masses were calculated using the Astra V software (Wyatt) as described previously (Colledge et al., 2011 (link)).
Dawn heleos 2 18 angle
Manufactured by Wyatt Technology
The Dawn HELEOS-II 18-angle is a multi-angle light scattering (MALS) instrument designed for the characterization of macromolecules and nanoparticles in solution. It is capable of measuring the molar mass, size, and conformation of samples using the principle of static light scattering.
Automatically generated - may contain errors
Lab products found in correlation
Optilab rex, by Wyatt Technology (3 mentions)
Astra 5 software, by Wyatt Technology (2 mentions)
Lc 20ad pump, by Shimadzu (1 mentions)
Astra 6, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Optilab t rex, by Wyatt Technology (1 mentions)
Superdex s75 10 300, by GE Healthcare (1 mentions)
Superdex 200 10 300 size exclusion column, by GE Healthcare (1 mentions)
Optilab rex, by Shimadzu (1 mentions)
5 protocols using dawn heleos 2 18 angle
1
Determining Oligomeric State of VapG
2
SEC-MALS Analysis of ThsA and ThsB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SEC-MALS analysis was performed on the Superdex 200 Increase 10/300 GL column (GE Healthcare) coupled with the DAWN HELEOS II (18-angle) and Optilab T-rEX instruments (Wyatt Technology). The column was equilibrated with buffer (200 mM NaCl, 2 mM DTT, 20 mM HEPES pH 7.5), after which ThsA (11 mg mL−1) and ThsB (24 mg mL−1) were loaded onto the column at a flow rate of 0.5 mL min−1 at 25 °C. Data were analyzed using ASTRA 6 software (Wyatt Technology).
3
Oligomeric State Analysis of VapD Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For determination of the oligomeric state of VapD-core and VapD-full, the proteins were analysed by SEC-MALLS. Samples of protein at concentrations of 2.5 and 4.0 mg ml−1 for VapD-core and VapD-full, respectively, were loaded onto a Superdex S75 10/300 gel-filtration column equilibrated at 0.5 ml min−1 with a mobile phase consisting of 50 mM Tris–HCl pH 8.0, 150 mM NaCl. The eluate was passed through an SPD20A UV–Vis detector, a Wyatt DAWN HELEOS II 18-angle light-scattering detector and a Wyatt Optilab rEX refractive-index monitor with the system driven by a Shimadzu HPLC system comprising an LC-20AD pump. The data were processed and molecular masses were calculated using the Astra V software (Wyatt) as described previously (Colledge et al., 2011 ▶ ).
4
Molecular Mass Determination by Light Scattering
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The masses in solution of FtsB (22–103) and FtsQ (58–276) both singly and in a complex were estimated using an online Dawn Heleos II 18 angle light scattering instrument (Wyatt Technologies) coupled to an Optilab rEX online refractive index detector (Wyatt Technologies). Protein samples (100 µl) were resolved on a Superdex S-75 10/300 analytic gel filtration column (GE Healthcare), preequilibrated with 25 mM Tris (pH 7.4), 100 mM NaCl, and 1 mM DTT at 0.5 ml/min. Protein concentration was determined from the excess differential refractive index based on dn/dc (slope of refractive index against concentration) of 0.186 mg/ml. The light scattering and protein concentration at each point across the peaks in the chromatograph were used to determine the absolute molecular mass from the intercept of the Debye plot using Zimm’s model as implemented in the ASTRA version 5.3.4.20 software (Wyatt Technologies). In order to determine the interdetector delay volumes, band broadening constants, and the detector intensity normalization constants for the instrument, we used bovine serum albumin (BSA) as a standard prior to sample measurements.
5
Oligomeric State Analysis of LAP-A Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified and frozen TbLAP-A, TcLAP-A, and LmLAP-A were thawed and diluted to concentrations of 0.1, 0.5, 1.0, and 3.0 mg ml−1, and additionally, TbLAP-A was diluted to 5 mg ml−1. The LAP-As were incubated at the respective concentrations for a minimum of 3 h at room temperature to allow potential concentration-dependent oligomeric state changes. The samples were injected automatically into a SEC-MALLS system (Wyatt Dawn HELEOS-II 18-angle light scattering detector and Wyatt Optilab rEX refractive index monitor linked to a Shimadzu high-performance liquid chromatography system comprising LC-20AD pump, a SIL-20A Autosampler, and an SPD20A UV/Vis detector) with a Superdex 200 10/300 size exclusion column (GE Healthcare) and a running buffer containing 10 mM Tris (pH 8.0), 200 mM NaCl, and 1 mM DTT. The data were analyzed with the ASTRA software (version 5.3.4.14), and the molecular mass of each sample was calculated.
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!