Y2h gold yeast cells

Manufactured by Takara Bio

Sourced in United States

The Y2H Gold yeast cells are a genetically engineered strain of Saccharomyces cerevisiae designed for use in yeast two-hybrid (Y2H) assays. They are intended to facilitate the detection and analysis of protein-protein interactions.

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Lab products found in correlation

In fusion pcr cloning system, by Takara Bio (2 mentions) Clonexpressii one step cloning kits, by Vazyme (1 mentions) Quick easy yeast transformation mix kit, by Takara Bio (1 mentions) Pgbkt7 vector, by Takara Bio (1 mentions) Tcs sp8 se, by Leica (1 mentions) Pgad424 and pgbt9 vectors, by Takara Bio (1 mentions) Pdest gbkt7, by Thermo Fisher Scientific (1 mentions) Lr reaction, by Thermo Fisher Scientific (1 mentions) Two hybrid system 3, by Takara Bio (1 mentions) Gateway system, by Thermo Fisher Scientific (1 mentions) Pgbkt7, by Thermo Fisher Scientific (1 mentions) Pgadt7, by Takara Bio (1 mentions) Pgbkt7, by Takara Bio (1 mentions) Yeast strain y2h, by Takara Bio (1 mentions)

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Y2H Gold cells

8 protocols using y2h gold yeast cells

RcNAC72 and RcDREB2A Interaction Assay

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According to the instructions of ClonExpressII One Step Cloning Kits (Vazyme, Nanjing, China), the full length, N-terminal (1–526 bp) and C-terminal (527–1059 bp) of the RcNAC72 gene were inserted into the EcoRI and BamHI of the pGBKT7 vector. The recombinant plasmids and pGADT7 plasmid were transferred into Y2HGold yeast cells (Huayueyang, Beijing, China), referring to the Quick Easy Yeast Transformation Mix kit instructions (Clontech, San Jose, CA, USA). The transformed yeast cells were diluted 10-fold with sterile water and 10 μL of the diluted solution was spotted on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade-x-α-gal media, respectively. These were cultured upside down at 30 °C for 3 days, and the yeast growth was observed.
The full length of RcDREB2A was inserted into pGBKT7 vector as a prey, and the full length of RcNAC72 was inserted into pGADT7 as a bait. As mentioned above, pGBKT7- RcDREB2A and pGADT7- RcNAC72 plasmids were jointly transferred into Y2H yeast. These yeast cells were observed on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade-x-α-gal selective media. The primers used above are listed in Table S1.

Jia X., Zeng Z., Lyu Y, & Zhao S. (2022). Drought-Responsive NAC Transcription Factor RcNAC72 Is Recognized by RcABF4, Interacts with RcDREB2A to Enhance Drought Tolerance in Arabidopsis. International Journal of Molecular Sciences, 23(3), 1755.

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Subcellular Localization and Transactivation Assay of CmNAC34

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The CDS of CmNAC34 missing stop codon was amplified and subcloned into the pCAMBIA1300 vector with a GFP to generate a 35S::CmNAC34-GFP fusion construct, and an empty pCAMBIA1300 vector was used as the positive control. These plasmids were introduced into tobacco leaves through Agrobacterium tumefaciens infiltration. Three days after the injection, tobacco leaves were collected, and the GFP fluorescence was observed using a laser confocal fluorescence microscope (TCS SP8-SE; 158 Leica, Wetzlar, Germany) after being stained by the nucleus marker DAPI.
For transcriptional activation assay, the CDS of CmNAC34 was subcloned into the pGBKT7 vector (Clontech, Mountain View, CA, USA) to produce pGBKT7-CmNAC34 fusion plasmid. The empty pGBKT7 vector and pGBKT7-53 + pGADT7-RecT were used as the negative and positive control, respectively. These plasmids were separately transformed into Y2H Gold yeast cells according to the manufacturer’s instructions (Clontech, Mountain View, CA, USA). The transformed yeast cell was cultured on amino acid-deficient medium SD/-Trp for 3–5 days. Then, several positive colonies were dotted on SD/-Trp-His-Ade with X-α-gal and incubated for 3–5 days at 30 °C to evaluate the transcriptional activity.

Zhao Y., Duan X., Wang L., Gao G., Xu C, & Qi H. (2022). Transcription Factor CmNAC34 Regulated CmLCYB-Mediated β-Carotene Accumulation during Oriental Melon Fruit Ripening. International Journal of Molecular Sciences, 23(17), 9805.

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Yeast Two-Hybrid Assay for Protein-Protein Interactions

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The pGAD424 and pGBT9 vectors (Clontech) were used to perform yeast two-hybrid (Y2H) assays. The pGAD424 vector contained a GAL4 activation domain, and the pGBT9 vector contained a GAL4 binding domain. The ORFs of the MdTCP46 and MdMYB1 sequences lacking the autonomously activated fragments were fused to the pGAD424 and pGBT9 vectors, respectively. MdTCP3-pGAD, MdTCP12-pGAD, MdTCP21-pGAD, MdTCP46-pGAD, MdMYB1^△-pGBD, MdBT2-pGBD, MdBT2-N-pGBD, and MdBT2-C-pGBD were constructed as described above. The genetic transformation of Y2H Gold yeast cells (Clontech) was accomplished by PEG induction. Transformed yeast cells were cultured on a selective medium for 3 d, and Y2H assays were performed as previously described (An et al., 2019b (link), 2020 (link)). The pGAD424 and pGBT9 empty vectors were used as negative controls.

An J.P., Liu Y.J., Zhang X.W., Bi S.Q., Wang X.F., You C.X, & Hao Y.J. (2020). Dynamic regulation of anthocyanin biosynthesis at different light intensities by the BT2-TCP46-MYB1 module in apple. Journal of Experimental Botany, 71(10), 3094-3109.

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Transactivation activity of CnTCP4 analyzed

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The transactivation activity of CnTCP4 was tested with a yeast one hybrid assay [48 (link)]. The three different C’-deletion variants of the CnTCP4 coding region (Figure 3c) were amplified using the primer pair CnTCP4-pENTR1A-F plus one of CnTCP4-pENTR1A-R1/-R2/-R3 (sequences given in Table S1). The resulting fragments were inserted into pENTR™1A via the BamHI and NotI cloning sites. The resulting pENTR™1A-CnTCP4, pENTR™1A-CnTCP4-F1, pENTR™1A-CnTCP4-F2, and pENTR™1A-CnTCP4-F3 fusions were subsequently recombined with pDEST-GBKT7 via an LR reaction (Invitrogen) to form the constructs pDEST-GBKT7-CnTCP4, pDEST-GBKT7-CnTCP4-F1, pDEST-GBKT7-CnTCP4-F2, and pDEST-GBKT7-CnTCP4-F3. Each of these four constructs, plus pCL1 (positive control) and pDEST-GBKT7 (negative control), were introduced individually into Y2H Gold yeast cells (Clontech, Mountain View, CA, USA) following the manufacturer’s protocol. Selection for transformants (except for those carrying pCL1) was carried out by culturing the cells on SD/-Trp medium, while the pCL1 transformants were cultured on SD/-Leu medium. All of the transformant cell lines were finally plated on SD/-His/-Ade medium containing 20 mg/mL X-α-Gal, and incubated at 30 °C.

Qi X., Qu Y., Gao R., Jiang J., Fang W., Guan Z., Zhang F., Zhao S., Chen S., Chen F, & Wang H. (2019). The Heterologous Expression of a Chrysanthemum nankingense TCP Transcription Factor Blocks Cell Division in Yeast and Arabidopsis thaliana. International Journal of Molecular Sciences, 20(19), 4848.

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Yeast Two-Hybrid Screening of Anther cDNA Library

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The yeast cDNA library of the anther tissue was introduced into the yeast strain Y187. The analyses were performed according the manufacturer’s instructions (Clontech, USA). The transcription activation activity of the bait protein was analyzed through the yeast growth status and an α-galactosidase activity assay on medium SD (−Trp/−Ade/−His) plates. For yeast cDNA library screening, Y2H colonies containing the pGBKT7-TaICK1 vector were mated with yeast strain Y187 (cDNA library of Anthers tissue), according to the instructions of the Two-Hybrid System 3 (Clontech, USA). Then, the mating type was selected on a high-stringency medium SD (−Ade/−His/−Leu/−Trp). Finally, after cloning the full length CDS of the prey gene, the prey genes were reconstructed into pGADT7. To further verify the protein interactions, the pGBKT7-bait vector and pGADT7-prey vector were co-transformed into Y2H Gold yeast cells (yeast strain Y2H, Clontech, USA), and the positive clones were selected on the SD medium (−Leu/−Trp). Positive yeast clones were picked and spread on the SD medium (−Ade/−His/−Leu/−Trp/X-α-Gal) to assay for protein interactions.

Zhang L., Wang C., Yu Y., Zhang Y., Song Y., Li Z., Wang S., Zhang Y., Guo X., Liu D., Li Z., Ma S., Zheng J., Zhao H, & Zhang G. (2020). Cyclin-Dependent Kinase Inhibitor Gene TaICK1 acts as a Potential Contributor to Wheat Male Sterility induced by a Chemical Hybridizing Agent. International Journal of Molecular Sciences, 21(7), 2468.

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Yeast Two-Hybrid Analysis of IbNAC3 Interactions

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The full-length coding sequence of IbNAC3 was inserted into the prey vector pGADT7 (Clontech) and the fulllength coding sequence of ANAC011, ANAC072, ANAC083, ANAC100, and NAP was separately cloned into the bait vector pGBKT7 (Clontech) by Gateway system (Invitrogen). Then the negative control, positive control, and indicated co-transformed recombinant constructs were introduced into the Y2HGold yeast cells according to the manufacturer's instructions (Clontech). A series of dilutions were transferred onto DDO (SD/-Trp-Leu), QDO (SD/-Trp-Leu-His-Ade), QDO/Aba (QDO with 200 ng/mL Aba), QDO/3-AT (QDO with 2 mM 3-AT), and QDO/Aba/ABA (QDO/Aba with 20 μM ABA) medium and were incubated at 30°C for 3-5 days to detect their survival.
For additional Y2H assays to map the protein domain of IbNAC3 that are required for each specific interaction with ANAC011, ANAC072, ANAC083, ANAC100, and NAP, all the truncated IbNAC3 sequences were amplified by PCR by the indicated primers and inserted into the pGBKT7 vector by Gateway system (Invitrogen). Different combinations of the recombinant pGADT7 and pGBKT7 plasmids were co-transformed into the yeast strain Y2HGold and the interactions were detected as above. Each interaction had at least three biological replicates with similar results, related primers were listed in Supplemental Table S6.

Meng X., Liu S., Zhang C., He J., Ma D., Wang X., Dong T., Guo F., Cai J., Long T., Li Z, & Zhu M. (2023). The unique sweet potato NAC transcription factor IbNAC3 modulates combined salt and drought stresses. Plant physiology, 191(1).

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Investigating ScDREB10 Transcriptional Activity

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To investigate the transcriptional activity of ScDREB10, the yeast two-hybrid system (Y2H) with pGBKT7 vector and Y2H yeast strain were used (Clontech). The coding sequence of ScDERB10 was fused in frame with the GAL4 DNA-binding domain (pGBKT7 vector) to produce the fusion construct of BD-ScDREB10 using the in-fusion PCR cloning system (Clontech); the full-length ORF of ScDREB10 was amplified from pMD18-T plasmids by PCR using gene-specific primers containing EcoR I and BamH I restriction sites, and the PCR product was inserted into the EcoR I/BamH I pGBKT7 vector. The construct was subsequently introduced into yeast Y2H Gold cells (Clontech). The yeast positive transformants were adjusted to an OD600 of 2.0, and the yeast cells were then 10-fold serially diluted and dropped with 2 µL on synthetic dropout (SD) medium without tryptophan (SD/−Trp), without tryptophan and histidine (SD/−Trp−His), and with SD/−Trp−His plates containing x-α-gal (5-Bromo-4-chloro-3-indolyl α-d-galactopyranoside) (SD/−Trp−His + x-α-gal). Yeast cells expressing containing the pGBKT7 empty vector or expressing GAL4 were used as the negative and positive control, respectively. The plates were incubated at 30 °C for 2 to 4 days before photographing. The primer information was listed in Table S1.

Li X., Liang Y., Gao B., Mijiti M., Bozorov T.A., Yang H., Zhang D, & Wood A.J. (2019). ScDREB10, an A-5c type of DREB Gene of the Desert Moss Syntrichia caninervis, Confers Osmotic and Salt Tolerances to Arabidopsis. Genes, 10(2), 146.

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Yeast Two-Hybrid Screening of ScDERB5

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The coding sequence of ScDERB5 was fused in frame with the GAL4 DNA-binding domain (pGBKT7 vector) to produce the BD-ScDREB5 fusion construct using the in-fusion PCR cloning system (Clontech), and the full-length ORF of ScDREB5 was amplified from pMD18-T plasmids by PCR using gene-specific primers containing EcoRI and BamHI restriction sites, and the PCR product was inserted into the EcoRI/BamHI pGBKT7 vector. The construct was subsequently introduced into yeast Y2H gold cells (Clontech). The yeast positive transformants were adjusted to an OD600 of 2.0, and the yeast cells were then serially diluted 10-fold and dropped with 2 μL of synthetic dropout (SD) medium without tryptophan (SD/-Trp), without tryptophan and histidine (SD/-Trp/-His), and with SD/-Trp/-His plates containing x-α-gal (SD/-Trp/-His + x-α-gal). Yeast cells expressing the pGBKT7 empty vector or expressing GAL4 were used as negative and positive controls, respectively. The plates were incubated at 30°C for 2–4 d before photographing. Primer information is listed in Supplementary Table 1.

Liu J., Yang R., Liang Y., Wang Y, & Li X. (2022). The DREB A-5 Transcription Factor ScDREB5 From Syntrichia caninervis Enhanced Salt Tolerance by Regulating Jasmonic Acid Biosynthesis in Transgenic Arabidopsis. Frontiers in Plant Science, 13, 857396.

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