Wild-type TG2, domain deletion mutants of TG2 (β-sandwich, core, β-sandwich-core) [41 (link)], the transamidase-deficient point mutant W241A [63 (link)], and the GTP-binding mutant R579A [40 (link)] were expressed as glutathione-S-transferase-TG2 fusion proteins in pGEX-2T expression vector (GE Healthcare, Sydney, NSW, Australia) in Escherichia coli M15 and purified as described previously [63 (link)]. TG2 used for animal experiments was dialyzed (6–8000 molecular weight cut-off) against sterile PBS (12 h, 4 °C). The protein was concentrated to 10 mg/mL with Ultragel 30K cut-off (Millipore, Burlington, MA, USA) and stored at 4 °C until use.
Pgex 2t expression vector
Manufactured by GE Healthcare
Sourced in Australia, United States
The PGEX-2T expression vector is a plasmid used for the expression of recombinant proteins in Escherichia coli. It contains the tac promoter for inducible expression and the glutathione S-transferase (GST) gene for affinity purification of the expressed proteins.
Automatically generated - may contain errors
Lab products found in correlation
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Ecori, by GE Healthcare (1 mentions)
Bamhi, by GE Healthcare (1 mentions)
Pgex 2t vector, by GE Healthcare (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Dna sequencing, by Eurofins (1 mentions)
4 protocols using pgex 2t expression vector
1
Purification and Characterization of Transglutaminase Variants
2
Mapping Epitope Binding of mAb5 on IRBP
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To determine which region of IRBP the mAb5 binds, we heterologously expressed the modules 1‐4 of bovine IRBP as fusion constructs with GST via pGEX2T expression vector (GE Healthcare, Chicago, IL, USA) and performed immunoblot assays of the purified protein. We constructed genes IRBPM1 (residues 1‐323), IRBPM2 (residues 320‐628), IRBPM3 (residues 625‐932), IRBPM4 (residues 930‐1286), IRBPM2_1 (residues 320‐366), IRBPM2_2 (residues 360‐488), IRBPM2_3 (residues 480‐575), IRBP M2_4 (residues 565‐630), IRBPM2_2A (residues 360‐416), and IRBP M2_2B (residues 410‐488) from the sequence of bovine IRBP and subcloned them into pGEX2T expression vector. All plasmids were transformed into the BL21 cell line (Sigma‐Aldrich, St. Louis, MO, USA) and cultured in 18.0 mL LB broth at 37°C in the presence of 2% glucose to suppress basal expression of GST from the lac promoter. When the culture OD600nm = 0.6 expression was induced with 1 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG, Sigma‐Aldrich) for 6 hours. Cultures were then centrifuged at 10 000 g for 30 minutes. GST fusion proteins were purified using the GSTrap system (GE Healthcare, Chicago, IL, USA) with some constructs requiring SP‐200 gel filtration to remove impurities. Samples (approximately 500 ng each) were subjected to SDS‐PAGE and silver‐stained or probed by immunoblot with anti‐GST or anti‐IRBP mAb5.
3
Recombinant Nf2/merlin and LATS1 Constructs
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We used our previously described Nf2/merlin construct, residues 1–339 [11] , which was cloned into the pGEX-2T expression vector (GE Life Sciences) by using the full-length human Nf2/merlin plasmid (Addgene plasmid number #11629). The human Nf2/merlin full-length construct, residues 1–595, was cloned into a pET-32a expression vector (Novagen) with a hexahistidine tag followed by a thioredoxin tag. LATS1 (residues 69–91) was synthesized by GenScript USA Inc.
4
Cloning and Expression of MRP6 L0 Domain
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The E 205 -G 279 region of human MRP6 protein was selected following bioinformatics analysis since it represents the L0 sequence with greater homology with the same domain of other MRP proteins.
The polypeptide was amplified by PCR from the expression vector pcDNA3.1-Flag-ABCC6 [8] (link) and cloned in pGEX-2T vector (GE Healthcare) using BamHI and EcoRI restriction enzymes. The forward primer 5′-TCGCGGATCCGAGACTGGGGCAGCCTTCC-3′ and the reverse primer 5′-CGAGGTACCGAATTCTCAGCCACTGCCGCCTTTC-3′ were used. For amplification GoTaq® qPCR Master Mix (Promega) and 30 ng of template were used in a 25 μL reaction volume. PCR reaction mixture was subjected to an initial denaturation of 2 min at 95 °C, 30 cycles of 1 min denaturation at 95 °C, 1 min annealing at 59 °C and 30 s amplification at 72 °C and a final amplification step of 5 min at 72 °C. The PCR product was revealed on a 1.5% agarose gel and purified by QIAquick PCR purification kit (Qiagen).
L0 construct was designed as a fusion GST-tagged protein. The L0 fragment and the pGEX-2T expression vector (GE Healthcare Life Sciences) were digested with EcoRI (BioLabs) and BamHI (BioLabs) restriction enzymes and then ligated. After ligation, One Shot Top Ten cells (Invitrogen) were transformed with the ligation product. The sequence and the correct orientation of the insert were confirmed by DNA sequencing (Eurofins Genomic, Germany).
The polypeptide was amplified by PCR from the expression vector pcDNA3.1-Flag-ABCC6 [8] (link) and cloned in pGEX-2T vector (GE Healthcare) using BamHI and EcoRI restriction enzymes. The forward primer 5′-TCGCGGATCCGAGACTGGGGCAGCCTTCC-3′ and the reverse primer 5′-CGAGGTACCGAATTCTCAGCCACTGCCGCCTTTC-3′ were used. For amplification GoTaq® qPCR Master Mix (Promega) and 30 ng of template were used in a 25 μL reaction volume. PCR reaction mixture was subjected to an initial denaturation of 2 min at 95 °C, 30 cycles of 1 min denaturation at 95 °C, 1 min annealing at 59 °C and 30 s amplification at 72 °C and a final amplification step of 5 min at 72 °C. The PCR product was revealed on a 1.5% agarose gel and purified by QIAquick PCR purification kit (Qiagen).
L0 construct was designed as a fusion GST-tagged protein. The L0 fragment and the pGEX-2T expression vector (GE Healthcare Life Sciences) were digested with EcoRI (BioLabs) and BamHI (BioLabs) restriction enzymes and then ligated. After ligation, One Shot Top Ten cells (Invitrogen) were transformed with the ligation product. The sequence and the correct orientation of the insert were confirmed by DNA sequencing (Eurofins Genomic, Germany).
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