The expression vector encoding p23 was derived from pGEX-6p-1 (GE Healthcare), and the proteins were produced in Escherichia coli BL21(DE3) cells grown at 20°C and induced with 1 mM isopropyl β-d -thiogalactoside. Cells were separated from the broth by centrifugation, resuspended in lysis buffer [25 mM tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 1 mM TCEP-HCl, and 1 × protease inhibitor cocktail (PIC)] and subjected to homogenization using a high-pressure cell disruptor. After centrifugation at 15,000g and 4°C for 1 hour to remove cell debris, the supernatant (cleared lysate) was incubated with GSH-Sepharose beads or amylose agarose beads (NEB) [column volume (CV): 2.5 ml] under shaking at 4°C overnight. The beads were loaded onto a gravity column, and the flow-through was collected. The column was washed with 30 CV (75 ml) (10 CV per drip wash, three washes) of wash buffer (lysis buffer without PIC) and eluted with 10 CV (25 ml) of elution buffer [lysis buffer with 20 mM d -(+)-maltose, without PIC].
Amylose agarose beads
Manufactured by New England Biolabs
Sourced in United States
Amylose agarose beads are a laboratory product used for affinity chromatography. They consist of agarose beads with covalently attached amylose, a polysaccharide derived from starch. The beads can be used to purify proteins that have a specific affinity for amylose, such as proteins with a maltose-binding protein (MBP) tag. The amylose agarose beads provide a efficient method for isolating and purifying these target proteins from complex mixtures.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pgex 6p 1, by GE Healthcare (1 mentions)
E coli rosetta de3 cells, by Transgene (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Glass bead, by Merck Group (1 mentions)
E coli bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
Pmal c5x, by New England Biolabs (1 mentions)
Halt protease inhibitor, by Merck Group (1 mentions)
Bca protein quantitation kit, by Thermo Fisher Scientific (1 mentions)
Pmal protein fusion and purification system, by New England Biolabs (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Myiq real time system, by Bio-Rad (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Dnase 1, by Qiagen (1 mentions)
5 protocols using amylose agarose beads
1
Purification of p23 Fusion Proteins
2
Purification of MBP-tagged Photosynthesis Proteins
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Full-length genes of Fd, NdhV, or NdhO from T. elongatus BP-1 (NIES-2133) and Synechocystis 6803 (ATCC 27184) were cloned into the pMAL vector, which has an N-terminal fused MBP tag and transformed into the E. coli Rosetta (DE3) cells (TransGen Biotech Co., Ltd). The cells were cultured in Luria broth at 37 °C until the OD600 reached about 1.0 and were then induced with 0.1 mM isopropyl-beta-d -thiogalactopyranoside and allowed to grow at 18 °C overnight. The cells were harvested by centrifugation and the pellets were resuspended in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol). The cells were then lysed by sonication and the cell debris was removed by ultracentrifugation. The supernatant was mixed with amylose agarose beads (New England Biolabs) and rocked for 4 h at 4 °C before elution with 20 mM maltose. The PreScission protease was added to remove the MBP tag. The proteins were then further purified by Mono-Q and by gel filtration chromatography equilibrated with 25 mM Tris, pH 6.5, and 100 mM NaCl. The purified proteins were concentrated to 30 mg mL−1 and stored at −80 °C.
3
Purification and Interaction of Oct4 and Trib2
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His-tagged Oct4 and Trib2 maltose-binding protein (MBP) fusion proteins were expressed in Escherichia coli and lysed in lysis buffer B (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.2% Triton X-100). Recombinant His-Oct4 and MBP-fused Trib2 proteins were purified by affinity chromatography on Ni-NTA agarose (Qiagen Inc., Valencia, CA, USA) or amylose agarose beads (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Purified His-tagged Oct4 and MBP-fused Trib2 proteins were incubated with pull-down buffer. After an additional 1 h of incubation, bound protein complexes were washed four times with binding buffer. The resulting protein complexes were eluted from the beads by boiling in 2 × SDS sample buffer, resolved on SDS-PAGE, and subjected to western blotting with anti-Trib2 antibodies.
4
Expression and Purification of Recombinant SmSoxS1
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Recombinant SmSoxS1 was produced, expressed, and purified as a Fusion protein with Maltose binding protein (MBP) using the pMAL Protein Fusion and Purification System (NEB, Ipswitch, MA, USA). SmSOXS1 was subcloned into pMAL-c5X (NEB, Ipswitch, MA, USA), and this plasmid construct was transformed and induced in BL21 (DE3) E. coli cells (Invitrogen, Waltham, MA, USA) with 0.4 mM IPTG. Cells were disrupted by sonication in lysis buffer (50 mM potassium phosphate pH 8.0, 200 mM sodium chloride; Halt protease inhibitor (Thermo Scientific, Waltham, MA, USA); PMSF). Cleared supernatant was incubated with amylose–agarose beads (NEB, Ipswitch, MA, USA) with agitation overnight at 4 °C. The fusion protein was eluted from the beads using a maltose elution solution (50 mM potassium phosphate pH 8.0, 200 mM sodium chloride, 10.4 mM maltose) and quantified using the BCA Protein Quantitation kit (Thermo Scientific, Waltham, MA, USA). Schistosomula samples were disrupted by bead beating with glass beads (Sigma, Burlington, VT, USA) and sonication in a Tris-HCl lysis Buffer (25 nM Tris-HCl pH 7.5, 1 mM DTT, 1X Halt protease inhibitor, 1 mM PMSF). Protein was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).
5
Quantifying gene expression and protein interactions
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Total RNA was extracted using the RNeasy plant mini kit (Qiagen), treated with DNase I (Qiagen) and reverse transcribed by using the SuperScript first-strand synthesis system (Invitrogen). qRT-PCR was performed by using the MyiQ real-time system (Bio-Rad) and the iQ SYBR-Green Supermix (Bio-Rad). Ubiquitin Conjugase (UBC) and ACTIN1 (ACT1) were used as internal controls for BR-related and light-related experiments, respectively. Primer sequences of genes tested in qRT-PCR were listed in Supplemental Table 1.
In Vitro Pull-Down and the Semi-In Vivo Pull-Down Assays GST-HY5, GST-HY5N, GST-HY5C and MBP-BZR1, MBP-BZR1N, MBP-BZR1C were respectively expressed in the Escherichia coli strain BL21 by induction with 0.3 mM isopropyl b-D-1-thiogalactopyranoside. The GST and MBP fusion proteins were purified by using glutathione-Sepharose beads (GE Healthcare) or amylose agarose beads (New England Biolabs), respectively. The in vitro pull-down assay of MBP-BZR1 and GST-HY5 interaction followed the procedure described previously (Li et al., 2012) .
For the semi-in vivo pull-down assay, protein extracts from 12-day-old 35S::mBZR1-Myc transgenic seedlings treated with or without BL
In Vitro Pull-Down and the Semi-In Vivo Pull-Down Assays GST-HY5, GST-HY5N, GST-HY5C and MBP-BZR1, MBP-BZR1N, MBP-BZR1C were respectively expressed in the Escherichia coli strain BL21 by induction with 0.3 mM isopropyl b-D-1-thiogalactopyranoside. The GST and MBP fusion proteins were purified by using glutathione-Sepharose beads (GE Healthcare) or amylose agarose beads (New England Biolabs), respectively. The in vitro pull-down assay of MBP-BZR1 and GST-HY5 interaction followed the procedure described previously (Li et al., 2012) .
For the semi-in vivo pull-down assay, protein extracts from 12-day-old 35S::mBZR1-Myc transgenic seedlings treated with or without BL
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