The CDS of C4 was cloned into pGBKT7 and the intracellular catalytic domain of CLV1 (704AA-967AA) was cloned into pGADT7. Yeast two-hybrid (Y2H) assays were performed using the Matchmaker GAL4-based Two-Hybrid System 3 (Clontech) according to the manufacturer’s instructions. Interactions were tested by stringent selection (SD/–Leu/–Trp/–His) supplied with 1 mM 3-amino-1,2,4-triazole (Sigma). Yeast cells with pGBKT7-p53 and pGADT7-T (Clontech) were used as positive controls; the yeast cells with pGBKT7-Lam and pGADT7-T (Clontech) were used as negative controls.
Pgadt7 t
Manufactured by Takara Bio
Sourced in United States
The PGADT7-T is a yeast expression vector used for the expression of foreign proteins in Saccharomyces cerevisiae. It contains the GAL1 promoter, which allows for the inducible expression of the target gene. The vector also includes the TRP1 selectable marker, which allows for selection of transformed yeast cells.
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Lab products found in correlation
Pgbkt7 lam, by Takara Bio (9 mentions)
Pgadt7, by Takara Bio (8 mentions)
Pgbkt7 53, by Takara Bio (7 mentions)
Pgbkt7 p53, by Takara Bio (6 mentions)
Pgbkt7, by Takara Bio (6 mentions)
Pgbkt7 vector, by Takara Bio (2 mentions)
3 amino 1 2 4 triazole, by Merck Group (1 mentions)
Matchmaker gal4 based two hybrid system 3, by Takara Bio (1 mentions)
Pgadt7 vector, by Takara Bio (1 mentions)
Ah109, by Takara Bio (1 mentions)
Y2hgold, by Takara Bio (1 mentions)
Sd leu trp plates, by Takara Bio (1 mentions)
Clontech yeast protocols handbook, by Takara Bio (1 mentions)
Saccharomyces cerevisiae y2hgold, by Takara Bio (1 mentions)
15 protocols using pgadt7 t
1
Yeast Two-Hybrid Assay for C4 and CLV1 Interaction
2
Yeast-based TH1 Interaction Assay
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The full-length TH1 coding region from the wild type and the mutants was amplified by PCR using primer BD-TH1F/R (see Supplementary Table S3 ) and cloned into the pGBKT7 vector (Clontech). The resultant constructs were co-transformed with the empty pGADT7 vector (Clontech) into yeast strain AH109 containing the GAL4-UAS-β-galactosidase reporter gene. The transformants were grown on the solid medium lacking leucine and tryptophan. The β-Galactosidase activity was detected using the Colony-lift Filter Assay according to the manufacturer’s user manual. For dimer formation assay, the full-length TH1 coding region from the wild type and the mutants was amplified by PCR using primer AD-TH1F/R (see Supplementary Table S3 ) and cloned into the pGADT7 vector. The resultant constructs were co-transformed with the pGBKT7 vector containing different TH1 alleles into yeast strain AH109. The transformants were grown on selective solid medium SD-Leu-Trp (DDO) or SD-Leu-Trp-His-Ade (QDO). Yeast colony co-transformed with pGBKT7-53 and pGADT7-T (Clontech) was used as positive control, while yeast colony co-transformed with pGBKT7-Lam and pGADT7-T (Clontech) was used as negative control.
3
Yeast Two-Hybrid Assay for Protein-Protein Interactions
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The yeast two-hybrid assay was carried out using a GAL4 DNA-binding domain-encoding bait vector (pGBKT7) and a GAL4 activation domain-encoding prey vector (pGADT7) in the yeast strain AH109, as described by Liu et al. (30 (link)). All fusion constructs were made as full-length fusions. The interaction between human lamin C bait fusion (pGBKT7-Lam) and simian virus 40 (SV40) prey fusion (pGADT7-T) (Clontech) was used as a negative control, and the interaction between murine p53 bait fusion (pGBKT7-53) and pGADT7-T served as a positive control.
4
Evaluating CmbHLH32 Homodimerization Activity
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Full length of CmbHLH32 was cloned and inserted into pGBKT7 and pGADT7 vectors (TaKaRa) to obtain pGBKT7-CmbHLH32 and pGADT7- CmbHLH32 vectors. The pGBKT7-CmbHLH32 was transformed into the yeast strain AH109 (TaKaRa) with pGADT7-T vector for transcriptional activation activity test. Homodimerizing transcriptional activation activity of CmbHLH32 used cotransformation of pGBKT7-CmbHLH32 and pGADT7- CmbHLH32 vectors. Cotransformation of pGBKT7-53 + pGADT7-T and pGBKT7-Lam + pGADT7-T were used as positive control and negative control respectively. The transformed yeast cells were streaked on SD/-Trp/-His/Ade/3-AT solid medium in different dilutions (10−1, 10−2 and 10−3). After 3-day incubation at 30 ℃, the transcriptional activation activity of CmbHLH32 were evaluated according to growth status of transformed yeast cells.
5
Yeast-based Two-Hybrid Screening
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The corresponding vectors were transformed into yeast strains. Full-length ZmSAP8 was cloned and ligated into the pGBKT7 vector (TaKaRa, Nanjing, China, Cat. No. 630489). Synthetic plasmids expressing each protein were transformed. The interaction of pGBKT7-53 with pGADT7-T (TaKaRa, Cat. No. 630442) was used as a positive control. Transcriptional activation was analyzed according to methods described in the literature [72 (link)]. Transformed cells were cultured on SD/-Trp and SD/-Trp/-His/-Ade plates. After 3–5 days of incubation at 30 °C, recombinant colonies were visualized.
6
Yeast Two-Hybrid Screening Protocol
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Saccharomyces cerevisiae strain AH109, control vectors pGADT7, pGBKT7, pGADT7-T, pGBKT7-lam, and pGBKT7-p53 were purchased from Clontech (Mountain View, CA, USA). Yeast strain AH109 was co-transformed with the combination of the pGADT7 and the pGBKT7 plasmids. Transformed yeast cells containing both plasmids were first grown on SD-minus Trp/Leu plates (DDO) to maintain the two plasmids and then were sub-cloned replica plated on SD-minus Trp/Leu/Ade/His plate (QDO).
7
Yeast Two-Hybrid Assay for MvfR-QslA Interaction
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Yeast two-hybrid experiment was performed using Matchmaker Gold Yeast System (Clontech) following the manufacturer’s instructions. Briefly, DNA fragments encoding QslA and different domains of MvfR were PCR amplified with primers listed in Supplementary Table S3 . The resulting qslA gene was cloned in the prey vector pGADT7. The coding sequence of MvfR and its divided fragments were fused in-frame with the GAL4 DNA-binding domain of the bait vector pGBKT7. Each bait/prey pair was co-transformed into the yeast strain AH109.
AH109 cells carrying the transformed plasmid were grown for 3 days at 30°C on agar plates with all essential amino acids except for tryptophan, leucine, and histidine (SD-Leu-Trp-His), and adenine (SD-Leu-Trp-His-Ade). The transformants co-transformed with plasmids pGBKT7-53 and pGADT7-T (Clontech) were used as positive controls (Iwabuchi et al., 1993 (link); Li and Fields, 1993 (link)). The strength of the protein interactions was judged by the growth of the colony on selective media following the manufacturer’s instructions.
AH109 cells carrying the transformed plasmid were grown for 3 days at 30°C on agar plates with all essential amino acids except for tryptophan, leucine, and histidine (SD-Leu-Trp-His), and adenine (SD-Leu-Trp-His-Ade). The transformants co-transformed with plasmids pGBKT7-53 and pGADT7-T (Clontech) were used as positive controls (Iwabuchi et al., 1993 (link); Li and Fields, 1993 (link)). The strength of the protein interactions was judged by the growth of the colony on selective media following the manufacturer’s instructions.
8
Yeast Transformation Assay for T. brucei 4G Homologs
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Yeast strain PJ69-4A was cultivated overnight at 30°C in YPD medium (Ammerman et al. 2012 (link)). Each transformation used 1 mL cell suspension washed and resuspended in 100 µL TE/100 mM lithium acetate buffer and incubated at RT for 15 min. The cells were centrifuged and resuspended in 360 µL transformation buffer (1 × TE/1 mM LiOAc; 50% PEG 8000 and 2 mg/mL boiled salmon sperm DNA), simultaneously transformed with GBK (tryp+) and GAD (leu+) plasmids expressing TbE5 and individual T. brucei 4G homologs, and incubated for 30 min at RT. Subsequently, the cells were incubated at 42°C for 20 min, and then spun down. The pellet was resuspended in 2 mL dropout medium (minimal medium minus tryptophan and leucine) and incubated overnight at 30°C. After dropout incubation the OD600 was checked and all cultures centrifuged, diluted to OD600 0.5 in dropout medium (minimal medium minus tryptophan, leucine, and histidine), applied on to 2% agar dropout medium plates containing 3-amino-1,2,4-triazole (3AT) in serial dilutions, and incubated at 30°C for 5 d. The positive control plates used plasmids pGADT7-T and pGBKT7-53 (CLONTECH Laboratories Inc.) for transformation.
9
Yeast Two-Hybrid Protein Interactions
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Protein interactions were analyzed using GAL4 fusion proteins in a yeast two-hybrid system. Saccharomyces cerevisiae strain AH109 and control vectors pGADT7, pGADT7-T, pGBKT7-p53, and pGBKT7-Lam were purchased from Clontech. The AH109 yeast strain was transformed with the bait plasmid pGBK-UL80.5 (in fusion with GAL4-BD) and subsequently transformed with selected expression clones pGAD-UBC9 and UBC9 deletion mutant series (in fusion with GAL4-AD). Positive clones were selected on a synthetic dropout medium that lacked four nutrients, including tryptophan, leucine, adenine, and histidine (QDO), and were tested for β-galactosidase activity.
10
Yeast Two-Hybrid Screening of DENV Proteins
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Human MMP-9 gene was subcloned in pGBKT7, and DENV NS1, E, and NS4A were subcloned in pGADT7 respectively. control vectors pGBKT7, pGADT7, pGBKT7-p53, pGADT7-T, and pGBKT7-lam, and some reagents were purchased from Clontech Laboratories. All experimental procedures were done following the Matchmaker Gold Yeast 2-Hybrid System User Manual. Briefly, yeast strain AH109 cells were co-transformed with plasmid pGADT7 and plasmid pGBKT7. Transformed yeast cells were grown in SD-minus Trp/Leu plates (DDO) for 24–48 h, and then subcloned replica plated on SD-minus Trp/Leu/Ade/His plate (QDO) for another 48–96 h.
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