For the Y2H analysis, the CDS of AtGL3, AtEGL3, RrGL3, and RrEGL3 was amplified and ligated into pGADT7 (AD) through BamHI and SacI double digestion sites. Full-length CDS of RrTTG1, AtTTG1, and RrGL1 was amplified and inserted into pGBKT7 (BD) at EcoRI and SmaI sites. Each combination of AD and BD plasmids was co-transformed into yeast strain AH109 separately using the LiAC/PEG method (Gietz and Schiestl, 2007 (link)). pGADT7-T was co-transformed with pGBKT7-53 as a positive control. pGADT7-T and pGBKT7-Lam were transformed as the negative controls (Supplementary Figure 1 ). All transformants were grown on SD-Trp-Leu (SD-LW), SD-Trp-Leu-Ade-His (SD-LWAH), and SD-LWAH-X-α-gal medium to detect the α-galactosidase activity of the yeast strains. The images were photographed at 5 day after incubation.
PGADT7
Manufactured by A&D Company
The PGADT7 is a laboratory equipment designed for performing genetic analysis and research. It is a compact, versatile instrument that can be utilized in various applications within the scientific community.
Automatically generated - may contain errors
Lab products found in correlation
PGBKT7, BD (15 mentions)
Matchmaker Gold Yeast Two-Hybrid System, Takara Bio (3 mentions)
PGBKT7, Takara Bio (2 mentions)
3-amino-1,2,4-triazole (3-AT;, Merck Group (1 mentions)
PGADT7, Takara Bio (1 mentions)
YeastmakerTM Yeast Transformation System 2, Takara Bio (1 mentions)
Frozen-EZ Yeast Transformation II Kit, Zymo Research (1 mentions)
17 protocols using PGADT7
1
Yeast Two-Hybrid Analysis of Transcription Factors
2
Yeast Two-Hybrid Analysis of OsCAF1 and OsCRS2
The coding sequences of OsCAF1 and OsCRS2 (Os01g0132800) were amplified using gene-specific primers. Full-length OsCAF1 and partial-length OsCAF1 were cloned into pGBKT7 (BD) to generate the BD-OsCAF1, BD-OsCAF1-N, BD-OsCAF1-M, BD-OsCAF1-C, BD-OsCAF1-C1, BD-OsCAF1-C2, BD-OsCAF1-C3, and BD-OsCAF1-C4 plasmids. In addition, full-length OsCRS2 was cloned into pGADT7 (AD) to generate the AD-OsCRS2 plasmid. The pairwise plasmid was co-transformed into the AH109 yeast strain and growth on SD-T/L in a 28 °C incubator. Next, the co-transformed yeast strains were transferred to SD-T/L/H/A for interaction analysis.
3
Yeast Two-Hybrid Analysis of FaCRY1-FaCOP1 Interaction
In Arabidopsis, previous works have shown that the C-terminal domain of AtCRY1 is an important region for the interaction between AtCRY1 and AtCOP1 through yeast two-hybrid (Y2H) assay (Wang et al., 2001 (link); Yang et al., 2001 (link)). Further, Holtkotte et al. (2017) (link) found that blue light enhanced the interaction intensity of AtCRY1 protein and AtCOP1 protein in yeast. To investigate the protein-protein interaction between FaCRY1 and FaCOP1, the Y2H assays were conducted as described by our previous study (Liu et al., 2022 (link)). The C terminal region (amino acids 479-673) of FaCRY1 was amplified and inserted into multiple cloning site (MCS) of bait vector pGBKT7 (BD). The full-length CDS of FaCOP1 was inserted into the MCS of prey vector pGADT7 (AD). The bait and prey constructs were co-transformed into yeast strain Y2HGold using the PEG/LiAc-based method. Following transformation, the yeast cells were spread onto synthetically defined medium (SD/-Trp-Leu) and incubated at 30 °C for 3 days. Then, 10 independent clones were selected and transferred to SD/-Trp-Leu-Ade-His medium supplemented with Aureobasidin A (100 ng/mL) and X-α-gal and cultured under blue light (50 μmol·m-2·s-1) for 4 days. All primers used for the vector construction are listed in Supplementary Table 1
4
Yeast Two-Hybrid Assay for Cotton Transcription Factors
The full-length cDNA of GhMYB1a, GhMYC1, and GhbHLH34 was fused to pGADT7 (AD) and pGBKT7 (Clontech). The constructs were co-transformed into yeast strain AH109 (Clontech). Synthetic drop-out media lacking tryptophan (SD/-Leu/-Trp) was used for selecting the transformed yeast strains at 28 °C for 3 d, which were then transferred and streaked onto synthetic drop-out media lacking tryptophan and histidine (SD/-Lue/-Trp/-His). 3-Amino-1,2,4-triazole (3-AT) (Sigma, USA) was added to the SD/-Lue/-Trp/-His media for the autoactivity assay of GhMYB1a.
5
Yeast Two-Hybrid Screening of Arabidopsis LP Interactors
Arabidopsis seven-day-old wild-type seedlings were transferred to an LP medium for 1, 3, 5, and 7 d and then harvested for LP yeast library construction. Briefly, it was constructed by extracting mRNA and cloning the cDNA into the vector pGADT7 (AD), followed by the transformation of the recombinant vector into the yeast strain Y187. The full-length coding sequence of PRU2 was cloned into the bait vector pGBKT7 (BD). The LP library was screened using yeast mating according to the Matchmaker Gold Yeast Two-Hybrid System manufacturer’s protocol (Clontech).
To confirm the protein–protein interactions, the full-length and truncated VOZ1, TCP8, RPL10, PAF1, CAB1, FSD1, and PHL3 were cloned into the vector pGADT7 (AD), co-transformed with pGBKT7-PRU2 into the yeast strain AH109 and plated on an SD/-Leu/-Trp/-His-selective medium (-LWH). To confirm the interaction between PRU2 and CK2αs or CK2βs, the full-length CK2αs or CK2βs was cloned into the vector pPR3-N, co-transformed with pBT3-SUC-PRU2 into the yeast strain NMY51 and plated on an SD/-Leu/-Trp/-His/-Ade-selective medium (-LWHA). The primers used for the yeast two-hybrid system are list inTable S1 .
To confirm the protein–protein interactions, the full-length and truncated VOZ1, TCP8, RPL10, PAF1, CAB1, FSD1, and PHL3 were cloned into the vector pGADT7 (AD), co-transformed with pGBKT7-PRU2 into the yeast strain AH109 and plated on an SD/-Leu/-Trp/-His-selective medium (-LWH). To confirm the interaction between PRU2 and CK2αs or CK2βs, the full-length CK2αs or CK2βs was cloned into the vector pPR3-N, co-transformed with pBT3-SUC-PRU2 into the yeast strain NMY51 and plated on an SD/-Leu/-Trp/-His/-Ade-selective medium (-LWHA). The primers used for the yeast two-hybrid system are list in
6
Protein Structure Modeling and Yeast Two-Hybrid Assay
The protein sequences of OsS5H and mutated OsS5H were uploaded to the publicly accessible Phyre2 website (http://www.sbg.bio.ic.ac.uk/phyre2 ; Kelley et al., 2015 (link)) for modelling. Then protein models were further edited with ChimeraX software (Pettersen et al., 2021 (link)).
To perform Y2H assay, CDS of OsS5H2, OsS5H3, and OsS5H4 was constructed into vector pGBKT7 (BD), and truncated C‐terminus of OsUGT74H4 was cloned into pGADT7 (AD). Yeast strain AH109 was used for transformation, and the detailed experimental process was described in a report by Liu, Cai et al. (2020 (link)).
To perform Y2H assay, CDS of OsS5H2, OsS5H3, and OsS5H4 was constructed into vector pGBKT7 (BD), and truncated C‐terminus of OsUGT74H4 was cloned into pGADT7 (AD). Yeast strain AH109 was used for transformation, and the detailed experimental process was described in a report by Liu, Cai et al. (2020 (link)).
7
Investigating SLF-RNase Interactions
To further investigate the interaction between S2-LbSLFs and S-RNases, a yeast two-hybrid assay was employed. We used S2-LbSLFs as prey and S5-RNase and S2-RNase as bait. The S2-LbSLFs were divided into full-length SLFs, N-terminal SLFs, and C-terminal SLFs, which were then cloned and ligated into pGADT7(AD). The S2-RNase and S5-RNase in their full-length forms were ligated into pGBKT7(BD). Vectors containing different SLF fragments were co-transformed into the yeast Y2Hgold strain with S-RNase for interaction experiments. The transformed yeast cells were grown on -Leu/-Trp agar plates at 30 °C for 4 to 5 days. The colonies were further grown on -Leu/-Trp/-Ade/-His agar plates at 30 °C for an additional 3 to 4 days to test for interactions.
8
Yeast Two-Hybrid Screening for RhARF18 Interactors
A Y2H system was used to screen for RhARF18 interaction proteins in a cDNA library from rose floral buds (mixed from early stages 1–4). The coding region sequence of RhARF18 was constructed into pGBKT7 (BD) as bait. Alignment with the NCBI database revealed 52 potential interaction proteins, which are listed in Supplemental Table S3 . The CDS of RhHD2, RhHDA6, and RhHDA19 were constructed into pGADT7 (AD) as prey vectors. pGBKT7-53 and pGADT7-T were used as positive controls and pGBKT7-lam and pGADT7-T were used as negative controls. The bait and the prey vectors were co-transformed into yeast strain Y2H Gold. The transformants were then spotted onto SD/-Trp-Leu, SD/-Trp-Leu-His-Ade, SD/-Trp-Leu-His-Ade+Aureobasidin A and SD/-Trp-Leu-His-Ade+Aureobasidin A + X-gal. The primers used in Y2H assays are listed in Supplemental Table S6 .
9
Yeast Two-Hybrid Assay for eIF4E-FER Interaction
The gene expression levels were analyzed in ePlant (http://bar.utoronto. ca/eplant/) (Waese et al., 2017) . A heatmap was made with Morpheus (https://software.broadinstitute.org/morpheus/). Cluster analysis was also performed with Morpheus using the Euclidean distance metric.
Yeast Two-Hybrid Assay Y2H assays were performed as previously described (Chen et al., 2016; Du et al., 2016; Stegmann et al., 2017) . Briefly, the coding sequences of eIF4Es were cloned into pGADT7 (AD). Primers for cloning are shown in Supplemental Table 1. The kinase domain of FER was cloned into pGBKT7 (BD). Distinct plasmid pairs were transformed into yeast AH109 cells. The transformants were diluted and plated onto synthetic dropout Molecular Plant 13, 698-716, May 4 2020 ª The Author 2019. 711 RALF1-FERONIA Regulates Protein Synthesis Molecular Plant medium lacking tryptophan/leucine (+His) and synthetic dropout medium lacking tryptophan/leucine/histidine (ÀHis) but supplemented with 20 mM 3-amino-1,2,4-triazole for 7 days to test the interaction.
Yeast Two-Hybrid Assay Y2H assays were performed as previously described (Chen et al., 2016; Du et al., 2016; Stegmann et al., 2017) . Briefly, the coding sequences of eIF4Es were cloned into pGADT7 (AD). Primers for cloning are shown in Supplemental Table 1. The kinase domain of FER was cloned into pGBKT7 (BD). Distinct plasmid pairs were transformed into yeast AH109 cells. The transformants were diluted and plated onto synthetic dropout Molecular Plant 13, 698-716, May 4 2020 ª The Author 2019. 711 RALF1-FERONIA Regulates Protein Synthesis Molecular Plant medium lacking tryptophan/leucine (+His) and synthetic dropout medium lacking tryptophan/leucine/histidine (ÀHis) but supplemented with 20 mM 3-amino-1,2,4-triazole for 7 days to test the interaction.
10
Yeast Two-Hybrid Analysis of EcaICE1 and EcaHOS1
Yeast two-hybrid assays were carried out using the Matchmaker™ gold Yeast two-Hybrid Systems (Clontech, USA).
Different truncated CDS of EcaICE1 without transcriptional activation activity were subcloned into pGBKT7 (BD) to form the bait vector. The full-length CDS of EcaHOS1 was amplified and inserted into pGADT7 (AD) to construct the prey vector (AD-EcaHOS1). The primers are listed in Supplemental table S3. The bait and prey vectors were co-transformed into yeast strain gold Y2H using lithium acetate (LiAc) method. Then yeast cells were plated on SD/-LW medium (minimal media double dropouts, SD basal medium without Leu and Trp) according to the manufacturer's protocol (Clontech, USA) for 72 h. Transformed colonies were sprayed on SD/-LWHA medium (minimal media quadruple dropouts, SD medium with -Leu/-Trp/-Ade/-His) containing 125-μM Aureobasidin A (AbA), to test for possible protein-protein interactions, according to the yeast cell growth status. Each experiment replicated three technological repeats in separate experiments.
Different truncated CDS of EcaICE1 without transcriptional activation activity were subcloned into pGBKT7 (BD) to form the bait vector. The full-length CDS of EcaHOS1 was amplified and inserted into pGADT7 (AD) to construct the prey vector (AD-EcaHOS1). The primers are listed in Supplemental table S3. The bait and prey vectors were co-transformed into yeast strain gold Y2H using lithium acetate (LiAc) method. Then yeast cells were plated on SD/-LW medium (minimal media double dropouts, SD basal medium without Leu and Trp) according to the manufacturer's protocol (Clontech, USA) for 72 h. Transformed colonies were sprayed on SD/-LWHA medium (minimal media quadruple dropouts, SD medium with -Leu/-Trp/-Ade/-His) containing 125-μM Aureobasidin A (AbA), to test for possible protein-protein interactions, according to the yeast cell growth status. Each experiment replicated three technological repeats in separate experiments.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!