Using the Yeast Two-Hybrid System to Identify Protein–Protein Interactions - Plant Proteomics: Methods and Protocols - page 237

3. 3 M sodium acetate, pH 5.2 . Adjust the pH using diluted acetic
acid.
4. RIPA buffer. 2 mM sodium phosphate buffer, pH 7, 0.2 %
Triton X-100, 0.02 % SDS, 0.2 mM EDTA, pH 8, and 10 mM
sodium chloride. Store at room temperature.
5. Protease inhibitor cocktail (Sigma-Aldrich).
6. 100 mM phenylmethylsulfonyl fl uoride, PMSF , prepared in
methanol (Sigma-Aldrich).
7. 2 × Laemmli sample buffer. 10 % SDS, 20 % glycerol, 120 mM
Tris-HCl pH 6.8 and 0.02 % bromophenol blue. Store at
room temperature.
8. Acid-washed glass beads (425-600 μ m diameter

(Sigma-Aldrich)).

All solutions use ultrapure water and analytical grade reagents.

2.6 LacZ Assay

1. Z buffer: 0.1 M sodium phosphate monobasic, pH 7, 10 mM
potassium chloride, 1 mM magnesium sulfate. Adjust pH to
7.0 and autoclave. Prepare fresh when needed.
2. 4 mg/l o-nitrophenyl-beta- D -galactopyranoside . Dissolve
o-nitrophenyl-beta - D -galactopyranoside (Sigma-Aldrich) in
deionized water. Alternatively, a stock solution may be pre-
pared in phosphate buffer. When used as a substrate for
β

-galactosidase, a solution of 20.5 mg/ml is prepared in
100 mM sodium phosphate buffer, pH 7.3. The solution is
gently warmed to completely dissolve the product. Store in the
dark -20 °C.

3 Methods

In a GAL4 -based two-hybrid assay, a bait protein is expressed as a
fusion to the Gal4 DNA-binding domain (DNA-BD), while librar-
ies of prey proteins are expressed as fusions to the Gal4 activation
domain (AD).
Very few plant cDNA libraries are commercially available,
although there are companies that perform screening using pre-
made libraries or generate them using GAL4 or LexA-based sys-
tems. Libraries can be found through the following links:
http://www.arabidopsis.org/abrc/catalog/cdna_library1.html
http://www.hybrigenics-services.com/

3.1 Screening
a cDNA Library
for Interactors

Screening a cDNA library for interactors can be performed by
co-transformation or by mating. We include a protocol using co-
transformation in a GAL4 -based two-hybrid system. A protocol to
perform Y2H my mating using the GAL4 system is detailed in