Experiment Overview
| Repository ID: | FR-FCM-ZYUL | Experiment name: | qY2H 2017/07/06 | MIFlowCyt score: | 65.75% |
| Primary researcher: | David CLUET | PI/manager: | Martin Spichty | Uploaded by: | David CLUET |
| Experiment dates: | 2017-06-29 - 2017-07-06 | Dataset uploaded: | Jan 2019 | Last updated: | Feb 2020 |
| Keywords: | [quantitative liquid Yeast two-hybrid] [affinity ladder] [Protein Protein Interaction] [Fluorescent fusion proteins] | Manuscripts: | |||
| Organizations: |
CNRS and Ecole Normale Superieure de Lyon, Lyon, (France)
|
||||
| Purpose: | The objective is to perform a quantitative yeast 2-Hybrid analysis. Bait proteins are fused with Tag RFP. Prey proteins are fused with yEGFP. The reporter gene under the control of the LexA operator is a tandem of Tag BFP (pSB_BFP2 plasmid). An affinity ladder with dual gating on Tag RFP-H and yEGFP-H is performed. | ||||
| Conclusion: | All cumulative Tag BFP curves are ordered in agreement with the affinity of the various couples. This set permits to reproduce Figure 4 of our publication with additional couples. | ||||
| Comments: | Samples of 10 million cells were generated to determine the minimal number of cells required to obtain a converged affinity ladder. 1. Gating: Tag RFP-H: 700-900 (linear scale) 2. Gating: yEGFP-H: 5000-6000 (linear scale) | ||||
| Funding: | CNRS ENS fond Recherche | ||||
| Quality control: | FCS collection for software testing | Non fluorescent empty Bait and Prey couple (sample NF-NF.fcs) is used for the setup of the acquisition parameters. Background signal of the system is determined with the fluorescent empty Bait and Prey couple (sample 0-0.fcs), which serves as negative control for interaction. The BD-B112 is a functional "covalent" (infinite affinity, sample B112-H102A.fcs) transcription factor. It is used as positive control for interaction. | |||
Experiment variables
| Timepoints | |
|---|---|
| · 2h | 0-0.fcs · 29A-H102A.fcs · 29F-H102A.fcs · 38F-H102A.fcs · 39A-H102A.fcs · B112-H102A.fcs · Ras-CRaf A85K.fcs · Ras-CRaf.fcs · NF-NF.fcs |
| Sample Type | |
|---|---|
| · EGY42a x TB50alpha diploids | 0-0.fcs · 29A-H102A.fcs · 29F-H102A.fcs · 38F-H102A.fcs · 39A-H102A.fcs · B112-H102A.fcs · Ras-CRaf A85K.fcs · Ras-CRaf.fcs · NF-NF.fcs |
| Conditions | |
|---|---|
| · 100ml SGR-UHW with 0.25% Galactose and 1% Raffinose at 30°C | 0-0.fcs · 29A-H102A.fcs · 29F-H102A.fcs · 38F-H102A.fcs · 39A-H102A.fcs · B112-H102A.fcs · Ras-CRaf A85K.fcs · Ras-CRaf.fcs · NF-NF.fcs |
