Recombinant proteins were purified by HiTrap Protein A column (GE Healthcare) or anti-FLAG M2 affinity gel (Sigma). In some experiments, different protein fractions of size exclusion chromatography (SEC) by SEC650 column (BioRad) were collected, and then performed for a size exclusion chromatography with Multi-Angle Light Scattering analysis (SEC-MALS) for absolute molecular mass and size measurements using Agilent 1200 system (Agilent Technologies) equipped with Superdex 200 column (GE Healthcare). Protein interactions were measured and analyzed by an Octet Red instrument (PALL, NY). The Octet Protein A sensor was dipped into solution containing mouse or human LAG3-Ig fusion protein (10ug/ml) and subsequently loaded with various concentrations of FLAG tagged FGL1 proteins (two-fold serial dilutions) or their different SEC peak fractions (10ug/ml). Protein association and disassociation was monitored and analyzed by Octet software, and the Kd value was calculated using 1:1 binding ratio.
Sec 650 column
Manufactured by Bio-Rad
The SEC 650 column is a size-exclusion chromatography column designed for the separation and purification of biomolecules. It operates on the principle of molecular size, allowing the separation of molecules based on their hydrodynamic volume. The column is suitable for a range of applications, including the analysis of proteins, peptides, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ngc chromatography system, by Bio-Rad (2 mentions)
Hitrap protein a column, by GE Healthcare (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Agilent 1200 system, by Agilent Technologies (1 mentions)
Optilab eex system, by Wyatt Technology (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
Dynapro nanostar, by Wyatt Technology (1 mentions)
Minidawn treos detector, by Wyatt Technology (1 mentions)
Biologic duoflow chromatography system, by Bio-Rad (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
7 protocols using sec 650 column
1
Recombinant Protein Purification and Interaction Analysis
2
Characterization of UvrC Oligomeric States
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
20 μM UvrB was mixed with either 25 μM UvrA1 or 25 μM UvrC (wild-type and truncated constructs; Table S1) in binding buffer composed of 50 mM Tris–HCl pH 8.0, 300 mM NaCl, 5% glycerol and 2 mM β-ME and after 15 min incubation at room temperature, the mixes were separated by SEC on a SEC650 column (Bio-Rad) equilibrated in binding buffer. Elution fractions were analyzed on TGX stain-free SDS-PAGE gels (Bio-Rad) allowing the protein bands to be visualized directly after migration on a Chemidoc MP imager (Bio-Rad). The oligomeric states of UvrC, UvrC-Δ(HhH)2, UvrC-NEndo and UvrC-C were analyzed by SEC coupled to multi-angle laser light scattering (MALLS), dynamic light scattering (DLS) and refractometry (RI). SEC-MALLS experiments were performed with a Superdex 200 10/300 GL column equilibrated with either 50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol and 5 mM β-ME or 50 mM Tris pH 8.0, 1 M NaCl, 5% glycerol and 5 mM β-ME. 20 μl protein solution at 2–10 mg ml−1 was injected onto the column at 0.5 ml min−1. On-line MALLS detection was performed with a miniDAWN-TREOS detector (Wyatt), DLS was recorded with a DynaPro Nanostar and RI measurements were performed with an Optilab eEX system (Wyatt).
3
Biophysical Analysis of apoA-IV Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WT and DM apoA-IV recombinant proteins were purified by size exclusion chromatography on a BioRad SEC650 column equilibrated in PBS. Protein concentration and purity were quantified by A280 and SDS-PAGE analysis, respectively. CD spectral scans and thermal melts of WT and DM apoA-IV (concentrations ranging from 0.4 to 0.5 mg/mL) were acquired on a Jasco J-810 spectropolarimeter using a 1 mm quartz cuvette (Helma). CD wavelength scans were collected between 190 and 250 nm and averaged over five accumulations. Raw data was then converted to molar ellipticity (deg.cm2.dmol−1). Thermal denaturation assays were carried out at a single wavelength (209 nm) by monitoring the change in ellipticity as the temperature was raised from 20 °C to 90 °C at a rate of 5 °C/min. All thermal denaturation data were baseline corrected, normalized between 0 (folded) and 1 (unfolded) and were fit to a nonlinear biphasic sigmoidal curve in GraphPad (GraphPad Software, San Diego, CA, USA).
4
Molecular Mass Estimation of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The apparent molecular masses of p204 HINab, HINa, HINb and mutant proteins were estimated using a 24 ml ENrich SEC 650 column (10/300 GL, Bio-Rad). Briefly, protein samples or molecular mass standards were applied to a 24 ml column at a flow rate of 0.6 ml/min and eluted with PBS. The standard proteins (Sigma-Aldrich, USA) used in this assay were β-amylase (200.0 kDa), albumin (66.0 kDa), and carbonic anhydrase (29.0 kDa).
5
Heparin Binding Interactions of A1M
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant A1M was incubated with heparin, HS2, HS3, HS4, HS5 or HS6 in ratios 1:2 (w/w) in 20 mM Tris-HCl, pH 8.0, 0.15 M NaCl for 30 min at room temperature. A1M was thereafter separated using a size exclusion ENrich SEC 650 column coupled to an NGC chromatography system (Bio-Rad Laboratories). Elution was performed with 20 mM Tris-HCl, pH 8.0 through addition of increasing concentrations of NaCl (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M). The heparin binding ability of the mutant form A1M-C34S, where the cysteine in position 34 is substituted for a serine, was examined under identical conditions.
6
Protein Purification via SEC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis was carried
out using a BioLogic DuoFLow chromatography system (Bio-Rad) with
an Enrich SEC 650 column. The samples were made up to 250 μL
with PBS. The protein concentrations (∼20 μM) were used
to generate a signal of around 45–50 mAU. The mixtures (∼20
μM each component) were incubated on the bench top at least
15 min before injecting.
out using a BioLogic DuoFLow chromatography system (Bio-Rad) with
an Enrich SEC 650 column. The samples were made up to 250 μL
with PBS. The protein concentrations (∼20 μM) were used
to generate a signal of around 45–50 mAU. The mixtures (∼20
μM each component) were incubated on the bench top at least
15 min before injecting.
7
Quaternary Structure Analysis of LpThi5 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!