Jul 11, 2019
Protein interaction analysis of KaiC3 with various Kai homologs via yeast two-hybrid experiments (β-Galactosidase Assay)
- Anika Wiegard1,
- Christin Köbler2,
- Katsuaki Oyama3,
- Anja K. Dörrich4,
- Chihiro Azai5,3,
- Kazuki Terauchi5,3,
- Annegret Wilde2,
- Ilka Maria IM Axmann6
- 1Heinrich-Heine Universität Düsseldorf;
- 2Institute of Biology III, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;
- 3Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
- 4Institute for Microbiology and Molecular Biology, Justus-Liebig University, 35392 Giessen, Germany;
- 5College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan;
- 6Institute for Synthetic Microbiology, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University Duesseldorf, 40225 Duesseldorf, Germany
- CyanoWorld
External link: https://doi.org/10.1128/JB.00478-19
Protocol Citation: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria IM Axmann 2019. Protein interaction analysis of KaiC3 with various Kai homologs via yeast two-hybrid experiments (β-Galactosidase Assay). protocols.io https://dx.doi.org/10.17504/protocols.io.v7ve9n6
Manuscript citation:
Wiegard A, Köbler C, Oyama K, Dörrich AK, Azai C, Terauchi K, Wilde A, Axmann IM, Array. Journal of Bacteriology 202(4). doi: 10.1128/JB.00478-19
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 06, 2018
Last Modified: July 11, 2019
Protocol Integer ID: 18389
Abstract
This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on a color change using β-galactosidase activity.
Materials
List of Materials
- Yeast cells (Y190)
- Frozen-EZ Yeast Transformation II Kit (Zymo Research)
- Yeast Nitrogen Base without amino acids (Formedium, CYN0401)
- Drop-out mixture (-Leu -Trp; MP Biomedicals, 114520012)
- Adenine-hemisulfate (Sigma-Aldrich, A9126)
- Bacto Agar (BD Diagnostics, 214010)
- Filter papers (MN 615, Macherey-Nagel)
- X-β-Gal (Roth, 2315.X)
- D-Glucose (Roth, X997.2)
- Na2HPO4 (Roth, 4984.1)
- NaH2PO4 (Merck, 10049-21-5)
- KCl (Merck, 7447-40-7)
- MgSO4 (Roth, P027.1)
- Parafilm
Complete supplement medium (CSM)
| Components for CSM Agar | ||
| Yeast Nitrogen Base with ammonium /without amino acids | 6.7 g/L | |
| D-Glucose | 20 g/L | |
| Bacto Agar | 20 g/L | |
| Drop-Out-Mix (amino acid mixture) | 0.60-0.64 g/L | |
| Adenine Hemisulfate | 50 mg/L | |
| dd H2O |
- Autoclave 15 min, 121°C or filter sterilize before using
Z-Buffer
| Components Z-buffer | final conc. | Stock solution | 50 ml | |
| Na2HPO4 | 60 mM | 0.5 M | 6 ml | |
| NaH2PO4 | 40 mM | 0.5 M | 4 ml | |
| KCl | 10 mM | 1 M | 0.5 ml | |
| MgSO4 | 1 mM | 1 M | 0.05 ml | |
| 39.45 ml H2O |
- Check pH, if not 7.0 adjust with 200 mM Na2HPO4 or 200 mM NaH2PO4
- Can be prepared as either 1x or 5x Solution and autoclaved
- Note Mg will fall out of solution on autoclaving, redissolve once cool by shaking
X-Gal Solution
- Prepare a 20 mg/ml stock solution of X-Gal in N,N-dimethylformamide (DMF) or dimethylsulfoxide (DMSO)
- Store stock solution protected from light at -20°C. Solutions may be stored at -20°C for 6-12 months.
Preparation of buffers
Preparation of buffers
Prepare
- CSM ‑Leu ‑Trp agar (complete supplement mixture lacking leucine and tryptophan)
- Z-Buffer
- X-Gal solution
Transformation of yeast cells
Transformation of yeast cells
Perform the transformation of yeast cells according to manufacturer's guidelines using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and select transformed cells on complete supplement mixture lacking leucine and tryptophan (CSM ‑Leu ‑Trp) at 30 °C for 3–4 days.
Re-plating of colonies
Re-plating of colonies
Spot formed colonies on a second plate (CSM ‑Leu ‑Trp)
- Resuspend 3 colonies in 100 µl H2O
- Spot 5 µl on the master plate
- Wrap parafilm around the plate
- Incubate at 30°C for 2 days
β-galactosidase assay
β-galactosidase assay
Prepare Z-Buffer/X-Gal solution
- 5 ml for each plate (10 cm circula)
- 10 ml Z-Buffer + 27 µl β-Mercaptoethanol + 42 µl X-Gal solution
For each plate to be assayed, pre-soak sterile filters in a petri-dish
- Stack 3 filters in a petri-dish and add 5 ml Z buffer/X-Gal solution
- Store in a plastic bag to prevent evaporation
β-galactosidase activity
- Prepare a petri-dish with 1 sterile and dry filter and mark the orientation of the filter (e.g. the top with a pencil line)
- Using a pair of tweezers place the dry filter in the correct orientation onto the plate with the colonies. Gently rub the filter with a sterile glass spreader to mediate clinging of the colonies to the filter (the filter becomes slowly wet) until spots are visible for all colonies
- Carefully lift the filter off the agar plate and transfer it to a pool of liquid nitrogen. Submerge the filter for ≈ 10 s
- After the filter is frozen completely replace it to the petri dish (colonies facing up) and let it thaw at RT
- Repeat freezing and defreezing for 3-5 times
- Remove excess liquid from the pre-soaked 3 stack filters
- Place the filter (colony side up) on the pre-soaked stack
- Avoid trapping air bubbles under or between the filters
- Colonies are supposed to look glossy
- Store the plates in a plastic bag and incubate at RT (if possible in the dark, since X-Gal is light sensitive)
- Check periodically for the appearance of blue colonies
- To stop the reaction remove the filter from the pre-soaked stack and let it dry open under the fume hood
- Scan the plates to record the results
- Result: Interaction of two proteins is positive if there is a blue color change of the respective colony, but without any color change of the controls