CG2712 [1–90] was cloned in frame with a TEV-cleavable GST-tag in the modified vector pGEX-4T1 (GE Healthcare). For in vitro experiments, protein fragments were PCR-amplified using corresponding primers (Supplementary Table S1 ) from fly cDNA and subcloned into modified pGEX-4T1 (GE Healthcare), pMAL-C5X (New England Biolabs), or a vector derived from pACYC and pET28a(+) (Novagen) bearing a p15A replication origin, kanamycin resistance gene, and pET28a(+) MCS. For yeast two-hybrid assays, cDNAs encoding ZADs were amplified using the corresponding primers (see Supplementary Table S1 ) and fused with the DNA-binding or activation domain of GAL4 in the corresponding pGBT9 and pGAD424 vectors (Clontech). We also used a modified pGBT9 vector in which ZADs were cloned at the N-terminus of the GAL4 DNA-binding domain. PCR-directed mutagenesis was used to generate constructs with mutant ZADs using mutagenic primers (Supplementary Table S1 ).
Pgex 4t 1
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The PGEX-4T-1 is a laboratory equipment product designed for protein expression. It functions as a vector for the expression and purification of recombinant proteins in Escherichia coli (E. coli) host cells.
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Lab products found in correlation
Glutathione sepharose 4b, by GE Healthcare (14 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (7 mentions)
Pet28a, by Thermo Fisher Scientific (7 mentions)
Pac5.1 plasmid, by Thermo Fisher Scientific (5 mentions)
Superdex 200, by GE Healthcare (4 mentions)
Pcdna3, by Thermo Fisher Scientific (4 mentions)
Glutathione superflow resin, by Takara Bio (3 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Pet28a, by Merck Group (3 mentions)
Pmal c5x, by New England Biolabs (3 mentions)
Amylose resin, by New England Biolabs (3 mentions)
Pmal c2, by New England Biolabs (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Superfi dna polymerase, by Thermo Fisher Scientific (3 mentions)
Pmal c2x, by New England Biolabs (3 mentions)
Pgex 4t 3, by GE Healthcare (3 mentions)
Glutathione sepharose bead, by GE Healthcare (3 mentions)
Pgex 4t 1 vector, by GE Healthcare (3 mentions)
Pegfp c1, by Takara Bio (3 mentions)
217 protocols using pgex 4t 1
1
Cloning and Mutagenesis of ZAD Proteins
2
Silencing and Tagging of Vinculin
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Endogenous vinculin was silenced using pSUPER-shVIN that was generated by subcloning an oligo targeting the human vinculin sequence TTCAAGAGA into the retroviral vector pSUPER-RETRO-PURO (Oligoengine; Peng et al., 2010 (link)). pLEGFP-WT vinculin was generated by amplifying full-length chicken vinculin, ligating it into the pENTR-DTOPO and then cloning it into a pLEGFP-DEST vector using the Gateway cloning system (Invitrogen). pLEGFP-DEST was digested with HindIII and BamH1 and ligated into pLEGFP-C1 vector and expression driven by CMV promoter. pLEGFP-vinculin Y822F was prepared using site-specific mutagenesis to introduce the appropriate single amino acid substitution into pLEGFP-WT vinculin (Peng et al., 2010 (link)). Full-length Snail was cloned into the pQCXIP retroviral vector and expression driven by CMV promoter (Place et al., 2013 (link)). pGEX4T1-vinculin 811–881 were constructed by PCR amplifying corresponding residues of chick vinculin and subcloning this into pGEX4T1 (GE Healthcare). pGEX4T1-vinculin 811–881 Y822F were prepared by using site-specific mutagenesis to introduce a mutation resulting in the appropriate single amino acid substitution into pGEX4T1-vinculin 811–881.
3
Purification and Interaction of Spc105 and LaeA
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Heterologous expression and subsequent purification of Spc105 and LaeA proteins were conducted in E. coli BL21 (λDE3) using a combination of GST fusion vector pGEX4T-1 (GE Healthcare) and N-terminal 6X histidine-tag fusion vector pET28a. Briefly, the spc105 gene was inserted into pGEX4T-1 to express GST-Spc105, which was subsequently purified on glutathione-agarose 4B (GE Healthcare) following manufacturer’s recommendations. The laeA gene was cloned into pET28a to yield a His6-LaeA fusion protein, which was purified using Ni-NTA agarose (GE Healthcare).
GST pull-down experiments were performed according to the manufacturer’s recommendations. Briefly, 20 μg purified GST-Spc105 protein, 25 μl glutathione magnetic beads (Pierce), and 20 μg purified His6-LaeA protein were co-incubated for 3 h at 4°C in PBS buffer. The magnetic beads were subsequently washed 6 times with PBS buffer and boiled for 10 min in SDS-PAGE loading sample buffer. SDS-PAGE and Western blot analysis were subsequently conducted. Immunodetection of His6-LaeA was performed using a Mouse His-tag monoclonal antibody at a dilution of 1:5000 (Proteintech) followed by Goat anti-Mouse HRP 1:10,000 (Proteintech).
GST pull-down experiments were performed according to the manufacturer’s recommendations. Briefly, 20 μg purified GST-Spc105 protein, 25 μl glutathione magnetic beads (Pierce), and 20 μg purified His6-LaeA protein were co-incubated for 3 h at 4°C in PBS buffer. The magnetic beads were subsequently washed 6 times with PBS buffer and boiled for 10 min in SDS-PAGE loading sample buffer. SDS-PAGE and Western blot analysis were subsequently conducted. Immunodetection of His6-LaeA was performed using a Mouse His-tag monoclonal antibody at a dilution of 1:5000 (Proteintech) followed by Goat anti-Mouse HRP 1:10,000 (Proteintech).
4
Cloning and Transfection of HDAC3 and Src Constructs
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Wild-type (WT) HDAC3 constructs were generated by standard PCR and cloned in the pSG5-KFM1-Myc, hemagglutinin (HA) (Sigma Aldrich, St. Louis, MO, USA), or pGEX4T-1 (GE healthcare, Piscataway, NJ, USA) vector.
The full length of c-Src or EGFR was also constructed by PCR and cloned into the pSG5-KFM1-Flag (Sigma Aldrich) vector. To generate HDAC3-CWT, the region from 277 aa to 428 aa of HDAC3 was amplified by PCR and subcloned in the pGEX4T-1 (GE healthcare) vector. HA-HDAC3-CY328/331A, shHDAC3-resistant Flag-HDAC3wt (Flag-rsh-HDAC3WT), and shHDAC3-resistant Flag-HDAC3Y328/331A (Flag-rshHDAC3Y328/331A) expression plasmids were derived from HA-HDAC3 or Flag-HDAC using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). All of the plasmid constructs were verified by DNA sequencing. Transient transfection was performed using TransIT 2020 (Mirus, Madison, WI, USA) according to the manufacturer’s instruction. To adequate transfection controls, the corresponding empty vectors were used in all of the relevant experiments.
The full length of c-Src or EGFR was also constructed by PCR and cloned into the pSG5-KFM1-Flag (Sigma Aldrich) vector. To generate HDAC3-CWT, the region from 277 aa to 428 aa of HDAC3 was amplified by PCR and subcloned in the pGEX4T-1 (GE healthcare) vector. HA-HDAC3-CY328/331A, shHDAC3-resistant Flag-HDAC3wt (Flag-rsh-HDAC3WT), and shHDAC3-resistant Flag-HDAC3Y328/331A (Flag-rshHDAC3Y328/331A) expression plasmids were derived from HA-HDAC3 or Flag-HDAC using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). All of the plasmid constructs were verified by DNA sequencing. Transient transfection was performed using TransIT 2020 (Mirus, Madison, WI, USA) according to the manufacturer’s instruction. To adequate transfection controls, the corresponding empty vectors were used in all of the relevant experiments.
5
Versatile Protein Expression Constructs
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New constructs were generated using standard polymerase chain reaction–ligation techniques and verified by automated sequencing. GST-tagged constructs were cloned into pGEX4T-1 (GE Healthcare) modified with a Tobacco etch virus (TEV) cleavage site and an N-terminal uncleavable His-tag (pGEX4T1 HG TEV) or pGEX4T-3 modified with a TEV cleavage site and a second ribosomal binding and multiple cloning site (pABLO TEV). Constructs with an N-terminal His-tag were cloned into pRSFDuet-1 (Novagen) modified with a TEV cleavage site following the N-terminal His-tag (pRSFDuet TEV) or into pRSFDuet TEV in which the N-terminal 6× His-tag has been replaced with a 12× His-tag (pRSFDuet 12× His TEV). DNA encoding the histone H3.1 tail (residues 1 to 43) was cloned into modified pRSFDuet-1 (Novagen), in which the N-terminal His-tag was deleted and the Xho I site was followed by a TEV cleavage site and GFP–His-tag. DNA encoding PARP1 and its variant was cloned into pET-21a(+) (Novagen) in-frame with the uncleavable C-terminal His-tag. For His-Ub, DNA encoding MHHHHHH-Ub was cloned into pET-3a (Novagen); for IR-Ub, DNA encoding GGSC-Ub was cloned into pGEX4T1 HG TEV. Mammalian constructs were cloned into pcDNA3 modified with an N-terminal Myc-tag (pcDNA3Myc), pGZdx21Z (28 (link)), pcDNA3.1 B(+), or mCherry2-C1, a gift from M. Davidson (Addgene plasmid no. 54563).
6
Cloning and Purification of NtCDPK1 Variants
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Plasmids pET16b-NtCDPK1 wild-type (WT), pGEX-4T-1-NtCDPK1 WT, S6A, T21A, S6A/T21A, D219N, and S6A/T21A/D219N, and pMALc2-RSG were previously generated [17 (link),29 (link)]. NtCDPK1 S6A/D219N and T21A/D219N were generated using overlap extension PCR and seamless ligation cloning extract (SLiCE) [30 (link)] with a plasmid harbouring full-length NtCDPK1 as a template and appropriate primers (S1 Table ). PCR products were cloned into pGEX-4T-1 (GE Healthcare). Glutathione S-transferase (GST)-NtCDPK1s, maltose-binding protein (MBP)-RSG, and 10×His-tagged (His)-NtCDPK1 were expressed in Escherichia coli harbouring pGEX-4T-1-NtCDPK1, pMALc2-RSG, or pET16b-NtCDPK1, respectively, and purified by glutathione-Sepharose 4B (GE Healthcare), Amylose Resin (New England Biolabs), or COSMOGEL His-Accept (Nacalai Tesque), respectively.
7
Constructing Expression Vectors for 4HAB56γ3
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All expression vectors for 4HAB56γ3 were constructed as reported previously [21 (link)]. pGEX4T-1-FLAG-p70S6K1 was prepared by excising cDNA encoding the FLAG-p70S6K from pcDNA3.1-FLAG-p70S6K1, which was subsequently cloned into the bacterial expression vector pGEX4T-1 (GE Healthcare). pLKO.1 vector harboring cDNA encoding the B56γ3-specific shRNA (TRCN0000002495) and shLuc (TRCN00000072244) were obtained from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan). For PPP2R5C (B56γ) knockout, vectors of gRNAs targeting PPP2R5C, pRP[CRISPR]-PPP2R5C[gRNA#2] and pRP[CRISPR]-PPP2R5C[gRNA#3], were obtained from Vectorbuilder (Taiwan), and the gRNA#2 sequence was 5'-CTGTGATCACATTCCGATTA 3' and the gRNA#3 sequence was 5'-TACGGGAGCGGAATTTGACC-3’.
8
GST-Fusion Protein Expression and Purification
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All constructs were created using standard molecular biological techniques. All constructs used for protein expression and purification in this study were GST fusions expressed from pGEX-4T1 (GE Life Sciences). The native proteins without signal peptides were expressed in BL21(DE3) Gold competent cells (Novagen). The gene encoding the LtgA deletion mutant lacking the alpha helix 503(ATAREIAGKIGMD)513 was chemically synthesized by ProteoGenix. The synthesized ltgA alpha helix deletion gene was cloned into a GST-fusion pGEX-4T1 (GE Life Sciences) plasmid as described above. The expression of all constructs was induced with 0.6 mM IPTG at an optical density at 600 nm (OD600) of 0.7–0.8 and harvested after 4 hr of induction at 18°C. After glutathione-affinity chromatography and thrombin cleavage, proteins were purified to homogeneity by size-exclusion chromatography (Superdex-200, GE) in 50 mM HEPES (pH 7.4), 150 mM NaCl, and 1 mM BME. After gel filtration, the proteins were immediately used for crystallization or flash frozen in liquid nitrogen and stored at −80°C.
9
Molecular Cloning of RdRp and GdDnaJ
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To generate the pET-32a-RdRp and pGEX-4 T-1-GdDnaJ fusion constructs used for GST pull-down experiments, RdRp and GdDnaJ were amplified and cloned into the pGEX-4 T-1 (GE Healthcare Life Sciences) and pET-32a (Novagen) vectors, respectively. For co-immunoprecipitation experiments, PCR amplifications of RdRp and GdDnaJ were introduced in frame into the respective pcDNA3.1-Myc-His (Invitrogen) and pcDNA3.1-HA vectors (Invitrogen) to generate the pcDNA3.1-Myc-His-RdRp and pcDNA3.1-HA-GdDnaJ plasmids. For bimolecular fluorescence complementation (BiFC) assay, RdRp and GdDnaJ were inserted into pbJUN-HA-KN151 vector between NheI and PvuI sites and pbFOS-Myc-LC151 vector between NheI and KpnI sites to construct pbFos-RdRp and pbJun-GdDnaJ plasmids, respectively. All the primers used in this study were designed by Oligo 7 software and are listed in Additional file 1 : Table S1.
10
Cloning and Mutagenesis of CSNK2 Genes
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The open reading frames (ORFs) of CSNK2B (GenBank: NM_001320.7) and CSNK2A1 (GenBank: NM_177559.3) were cloned in pGEX4T1 (GE Healthcare) and pEGFP-C1. Identified variants of CSNK2B GenBank: NM_001320.7, c.94G>C, c.94G>A, and c.374G>C, were introduced in these plasmids by site-directed mutagenesis (Promega). Oligonucleotides sequences used to create variants of CSNK2B are shown in Table S2 . We obtained GFP-CTNNB1 plasmid as a donation from Prof. Carien Niessen (Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases [CECAD], University of Cologne). This plasmid contains the ORF of CTNNB1 (GenBank: NM_001904.4), which was excised and introduced into pGEX4T1 plasmid.
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