Yeast two-hybrid techniques were performed according to the yeast protocols handbook and the Matchmaker GAL4 Two-hybrid System 3 manual (both Clontech, Heidelberg, Germany). Direct interaction of two proteins was investigated by cotransformation of the respective plasmids in the yeast strain Y190, followed by selection of transformants on medium lacking Leu and Trp at 30°C for 3 days and subsequent transfer to medium lacking Leu, Trp and His (supplemented with 25 mM 3-amino-triazole) for growth selection and lacZ activity testing of interacting clones.
Matchmaker gal4 two hybrid system 3 manual
Manufactured by Takara Bio
Sourced in Germany, United States
The Matchmaker GAL4 Two-hybrid System 3 manual provides instructions for a protein-protein interaction detection and analysis system. The manual outlines the core functionality of the system, which involves the use of the GAL4 transcription factor to identify and study interactions between proteins of interest.
Automatically generated - may contain errors
9 protocols using matchmaker gal4 two hybrid system 3 manual
1
Yeast Two-Hybrid Protein Interaction Analysis
2
Yeast Two-Hybrid Screening of XopS Interactors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Y2H techniques were performed according to the yeast protocols handbook and the Matchmaker GAL4 Two-hybrid System 3 manual (both Clontech, Heidelberg, Germany) using the yeast reporter strains AH109 and Y187. To identify XopS interacting proteins, a GAL4 AD-domain tagged cDNA library from N. tabacum (Börnke, 2005 (link)) was screened as detailed in Üstün et al. (2013) (link).
3
Yeast Two-Hybrid Screening of Arabidopsis Chromatin Remodelers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full length coding sequences of SCRM, SCRM2, SPCH, MUTE, FAMA, BRM, BRD1, SWI3C, SWI3D, BRIP2, SWP73B, LUH, and HAC1 were amplified from Arabidopsis thaliana cDNA and cloned into pGAD-T7 or pGBK-T7 (Clontech). Combinations of bait end pray plasmids were then co-transformed into yeast strain AH109 and positive transformants were selected for by growth on synthetic dropout (SD) medium without leucine and tryptophane (-LT). Bait-prey interaction was tested by growth complementation assays on SD medium without leucine, tryptophane, histidine and adenine (-LTHA) as described in the Matchmaker™ GAL4 Two-Hybrid System 3 manual (Clontech). To overcome auto-activation from some of the constructs, 3-Amino-1,2,4-triazole (3-AT) was added to nutrient selection plates at different concentrations.
4
Yeast Two-Hybrid Assay for Small GTPases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse retina cDNA library was generated according to 'Mate&Plate' Library System User Manual (Clonetech), cloned into pGADT7 (short: pAD) and introduced into Saccharomyces cerevisiae Y187. Yeast techniques and two-hybrid methods were performed according to the Yeast Protocols Handbook and the Matchmaker GAL4 Two-Hybrid System 3 manual (Clontech) with S. cerevisiae AH109. Murine Arl3△ND129N (residues 17–182), Arl6△ND133N (residues 16–186), Arl2△ND128N (residues 17–184) and Arl13B (residue 20–278)were cloned into a Gateway compatible pBD-Gal4 vector (a kind gift from R. Roepman) and S. cerevisiae AH109 used as recipient for transformation.
5
Constructing Brachypodium Y2H Clones
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain cDNA suitable for creating Y2H-compatible Brachypodium genes, Bd21-3 seedlings were grown on ½-MS plates and the bottom 5 mm (developmental zone) of young true leaves were harvested, flash-frozen, and ground for RNA extraction using the RNeasy Plant MiniKit (Quiagen), followed by reverse transcription with SuperScript IV reverse transcriptase (Thermo Fisher) according to the manufacturer's instructions. Genes with stop codons were amplified using the primers in Supplemental Table 1 and cloned into pENTR/D-TOPO, followed by LR recombination with Gateway-compatible pGAD-T7 and pGBK-T7 (Clontech). For Y2H assays, bait and prey plasmids were transformed into the yeast strain AH109, followed by the selection of transformants and testing of pairwise interactions by growth complementation assays on nutrient selection media as described in the Matchmaker™ GAL4 Two-Hybrid System 3 manual (Clontech; Takara Bio USA, Inc, 2009 ). Three replicates per pairwise interaction were tested, and representative images were used for the figures. To compensate for the auto-activation of some constructs, plates containing 10–30 mM 3-amino-1,2,4-triazole (3-AT) were included in the interaction assays.
6
Yeast Two-Hybrid Screening of Arabidopsis SCRM Family
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full-length coding sequences of SCRM, SCRM2, SPCH, MUTE, FAMA, BRM, BRD1, SWI3C, SWI3D, BRIP2, SWP73B, LUH, and HAC1 were amplified from Arabidopsis thaliana cDNA and cloned into pGAD-T7 or pGBK-T7 (Clontech). Combinations of bait and prey plasmids were then cotransformed into yeast strain AH109, and positive transformants were selected for by growth on synthetic dropout (SD) medium without leucine and tryptophan (-LT). Bait-prey interaction was tested by growth complementation assays on SD medium without leucine, tryptophan, histidine and adenine (-LTHA) as described in the Matchmaker GAL4 Two-Hybrid System 3 manual (Clontech). To overcome autoactivation from some of the constructs, 3-amino-1,2,4-triazole (3-AT) was added to nutrient selection plates at different concentrations.
7
Assessing Transcription Factor Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before analyzing the interaction between MeGRXC15 and TGA transcription factors, an autonomous transactivation analysis was performed in yeast strain Y187. The MeGRXC15 was in frame fused to GAL4 BD (binding domain) in pGBKT7, and then transformed into yeast Y187. Because MeGRXC15 shows “autonomous transactivation” in yeast, a MeGRXC15 GSH binding site mutant MeGRXC15mP65G75 was produced by replacing P65AVFIGGILVG75 to A65AVFIGGILVA75. Next, for identification the interaction between MeGRXC15 and TGA transcription factors, a yeast two-hybrid assay has been performed in yeast strain Y187 based on the Matchmaker ™ GAL4 two-hybrid system 3 manual (Clontech). The MeGRXC15 GSH binding site mutant DNA construct MeGRXC15mP65G75:pGBKT7 was used as bait. The cDNA sequences of TGA transcription factors from Arabidopsis and cassava were introduced into the pGADT7, respectively in frame fused to GAL4 activation domain (AD). The MeGRXC15mP65G75:pGBKT7 and TGA:pGADT7 constructs were pairwise co-transformed into yeast strain Y187. The presence of transgenes was confirmed by growth on SD/ -Trp/−Leu plates. Interactions between two proteins were checked by examining β-galactosidase activity as the manual instructed.
8
Cultivation Protocols for Model Organisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli strain TOP10 (Invitrogen, Thermofischer, Carlsbad, CA, USA) was used for cloning. Growth conditions and media for the cultivation of E. coli followed the protocols described before (Sambrook et al., 1989) . Saccharomyces cerevisiae strain AH109 (Clontech/Takara Bio, Mountain View, CA, USA) was used for yeast two-hybrid interaction studies. S. cerevisiae cells were grown in minimal medium (SD) supplemented with the amino acids dropout-mix needed for selection, as described in the Clontech/Takara Bio Matchmaker TM GAL4 Two-Hybrid System 3 Manual (https://www.takar abio.com). U. maydis cells were grown in YEPSL (Brachmann et al., 2001) , CM (complete medium) supplemented with 1% glucose (CM-Glc) or 1% arabinose (CM-Ara), respectively (Holliday, 1974) at 28°C. Solid media contained 2% agar. The induction of hyphal growth in AB31 derivatives was done as previously described in Brachmann et al. (2001) . A. nidulans was grown in MM (minimal medium) (Hill & Kafer, 2001) (link). All U. maydis and A. nidulans strains used in this study are listed in Tables S4 andS5, respectively. Growth rate was measured with OD 600 from three biological replicates and technical duplicates hourly over a time span of 12 hr.
9
Yeast Two-Hybrid Interaction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For interaction studies in yeast, plasmids pGBKT7 and pGADT7 (Clontech/Takara Bio, Mountain View, CA, USA) harboring were used, following the protocols given in the Clontech/Takara Bio Matchmaker TM GAL4 Two-Hybrid System 3 Manual (https://www. takar abio.com). Plasmids for yeast two hybrid interaction assays are listed in Table S6.
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!