Truncated soluble and Glutathione S-transferase (GST) tagged human FcRn lacking the transmembrane domain (hFcRn-GST) was produced in HEK293E cells and purified on a GSTrap column (Cytiva Life Sciences)67 (link). Truncated soluble and biotinylated human and mouse FcRn forms lacking the transmembrane domain were acquired from Immunitrack Inc. Truncated soluble His6x tagged human FcRn lacking the transmembrane domain (hFcRn-His) was produced in a Baculovirus expression system and purified using a HisTrap HP column (Cytiva Life Sciences)68 (link),69 . The Baculovirus stock was a kind gift from Dr. Sally Ward (University of Southampton). Monomeric fractions of hFcRn-His were isolated by SEC using a Superdex 200 Increase 10/300 column (Cytiva Life Sciences) with an ÄKTA Avant 25 (Cytiva Life Sciences).
Gstrap column
Manufactured by Cytiva
The GSTrap column is a chromatography column designed for the purification of glutathione S-transferase (GST) fusion proteins. It utilizes immobilized glutathione to capture and purify GST-tagged proteins from complex samples.
Automatically generated - may contain errors
4 protocols using gstrap column
1
Purification of Soluble Human FcRn
2
Purification of GST-tagged Protein
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Following
a fresh transformation into BL21
DE3 cells, the cultures were inoculated and grown at 37 °C until
the O.D.600nm reached ∼0.5. The cells were cooled
to 20 °C, induced with 0.2 mM IPTG, and left to express overnight.
The cells were harvested with centrifugation and resuspended in the
lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 2 mM β-mercaptoethanol,
and 5 mM MgCl2) and lysed using a microfluidizer at 20,000
psi. The lysed cells were centrifuged at 48,000g,
filtered through a 5 μM filter, and loaded onto a 5 ml GSTrap
column (Cytiva) at 3 mL min–1 using a P960 peristaltic
pump. The column was washed with 50 ml of the lysis buffer before
eluting with the lysis buffer supplemented with 10 mM glutathione.
The eluted protein was subsequently passed through a Superdex S200
16/60 size-exclusion column equilibrated in 20 mM Tris pH 7.5, 150
mM NaCl, 1 mM DTT, and 5 mM MgCl2.
a fresh transformation into BL21
DE3 cells, the cultures were inoculated and grown at 37 °C until
the O.D.600nm reached ∼0.5. The cells were cooled
to 20 °C, induced with 0.2 mM IPTG, and left to express overnight.
The cells were harvested with centrifugation and resuspended in the
lysis buffer (50 mM Tris pH 7.5, 300 mM NaCl, 2 mM β-mercaptoethanol,
and 5 mM MgCl2) and lysed using a microfluidizer at 20,000
psi. The lysed cells were centrifuged at 48,000g,
filtered through a 5 μM filter, and loaded onto a 5 ml GSTrap
column (Cytiva) at 3 mL min–1 using a P960 peristaltic
pump. The column was washed with 50 ml of the lysis buffer before
eluting with the lysis buffer supplemented with 10 mM glutathione.
The eluted protein was subsequently passed through a Superdex S200
16/60 size-exclusion column equilibrated in 20 mM Tris pH 7.5, 150
mM NaCl, 1 mM DTT, and 5 mM MgCl2.
3
Expression and Purification of PAK4(300-591)
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A gene fragment encoding PAK4(300-591) was amplified using primers with BamHI and XhoI sites and cloned into a pGEX-6P1 vector. The L301A mutation was then generated by site directed mutagenesis. The protein was expressed in E. coli BL21(DE3)pLysS cells by inducing with 1.0 mM IPTG at OD600 = 0.4, grown overnight at 18 °C. The protein was purified on a GSTrap column (Cytiva) and the GST tag cleaved with PreScission protease (Cytiva). The protein was further purified using a 26/600 Superdex 75 pg column (Cytiva) in 50 mM Tris [pH 7.5], 150 mM NaCl, 10 mM DTT.
4
Structural Analysis of Glycogen Synthase
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Detailed methods are provided in the online version of this paper and include the following:
between the GYG1-tail and GYG globular domains is shown as a dashed gray line, and the glycogen chain is represented by a dotted black line. In the inactive state, all active sites are closed and GYG1 remains distant from the GYG1 tetramer (left). After transition to the ordered state, a GYG1 dimer associates with two protomers of GYG1 enabling handoff of the glycogen chain (center). Transition to the fully active state allows for elongation as GYG1 again moves distant from the GYS1 tetramer (right). Article was added (ratio 1:10 w/w) and incubated overnight at 4 C. After cleavage, the protein complex was concentrated and loaded into a HiLoad 26/60 Superdex 200 pre grade column (Cytiva) pre-coupled in line with a 5 mL GSTrap column (Cytiva), pre-equilibrated with buffer B (25 mM Tris pH 8, 150 mM NaCl, 1 mM TCEP).
between the GYG1-tail and GYG globular domains is shown as a dashed gray line, and the glycogen chain is represented by a dotted black line. In the inactive state, all active sites are closed and GYG1 remains distant from the GYG1 tetramer (left). After transition to the ordered state, a GYG1 dimer associates with two protomers of GYG1 enabling handoff of the glycogen chain (center). Transition to the fully active state allows for elongation as GYG1 again moves distant from the GYS1 tetramer (right). Article was added (ratio 1:10 w/w) and incubated overnight at 4 C. After cleavage, the protein complex was concentrated and loaded into a HiLoad 26/60 Superdex 200 pre grade column (Cytiva) pre-coupled in line with a 5 mL GSTrap column (Cytiva), pre-equilibrated with buffer B (25 mM Tris pH 8, 150 mM NaCl, 1 mM TCEP).
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