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Immunoprecipitation of yeast proteins in denaturing conditions for analysis of ubiquitylation - METHOD 1

Sébastien Léon and Rosine Haguenauer-Tsapis

Centre Nationale de la Recherche Scientifique, Paris, France

INTRODUCTION

The aim of this experiment is to immunoprecipitate proteins from yeast in denaturing conditions. After blotting of the immunoprecipitate with anti-Ubiquitin antibodies (ex. clone P4D1), this allows to verify the ubiquitylation status of a given protein. This protocol is adapted from Kragt et al 2005.

MATERIALS

IP dilution buffer - 50 mM Tris pH 7.5, 2 mM EDTA - 100 mM NaCl - 1.2 % Triton X-100 - 0.5 % BSA - Add PIC & NEM 20 mM

Buffer B1 - 50 mM Tris pH 7.5, 2 mM EDTA - 100 mM NaCl - 1 % Triton X-100 - 0.2 % SDS

Buffer B2 - 50 mM Tris pH 7.5 - 2 mM EDTA - 100 mM NaCl - 0.1 % Triton X-100 - 0.5 % SDS - 0.5 % sodium DOC

Buffer B3 - 50 mM Tris pH 7.5 - 2 mM EDTA - 500 mM NaCl - 0.1 % Triton X-100

Buffer B4 - 50 mM Tris pH 7.5, 2 mM EDTA - 100 mM NaCl

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METHODS

1. Starting from 20 ODs of cells in exponential phase: lysis with glass beads in 5 % TCA.

2. Resuspend precipitates in 200 μl of:

- 2 % SDS containing some bromphenol blue (just enough to indicate pH)

- protease inhibitors cocktail (PIC, Sigma)

- NEM (20 mM)

- MG-132 (100 µM)

3. Adjust the pH with Tris-Base 1M (a few µL - until the buffer turns blue), and boil the samples for 5 min at 95ºC.

4. Spin down to remove undissolved material (room temperature, 13,000 g, 5 min).

5. Add to the supernatant 800 μl IP dilution buffer, containing PIC and 20 mM NEM. Take an aliquot, add 1/4 vol of sample buffer (initial extract).

6. Add antibody against the protein to immunoprecipitate and mix for 1h at 4°C.

7. Add 50 μl Protein G-Sepharose (GammaBind, GE Healthcare) and mix for 1h at 4°C.

8. Spin down gently (bench microcentrifuge; 10 sec). Take an aliquot of the supernatant (unbound fraction) and treat as in step 5, to compare with the initial extract and see the IP efficiency

9. Wash the beads

- once with buffer B1 (1 mL)

- once with buffer B2 (1 mL)

- once with buffer B3 (1 mL)

- once with buffer B4 (1 mL)

10. Elute immunoprecipitate by boiling the beads in 50 µL sample buffer. Analyze by SDS-PAGE and immunoblotting. Anti-ubiquitin can be used to see ubiquitin conjugates (Anti-Ub P4D1, Anti-Ub(P4D1)-HRP, FK1/FK2, monoclonal or polyclonal depending on the antibody used for the immunoprecipitation). His-tagged, HA-tagged or myc-tagged ubiquitins and their corresponding antibodies are also good alternatives.

Notes

This protocol can be performed on any kind of sample (samples from differential centrifugations, gradients; etc.).

After TCA precipitation, incubate on ice for an hour (at least) and use for immunoprecipitation as described above.

BIBLIOGRAPHY

Kragt, A., T.M. Voorn-Brouwer, M. Van den Berg, and B. Distel. 2005. The Saccharomyces cerevisiae peroxisomal import receptor Pex5p is monoubiquitinated in wild type cells. J Biol Chem. 280:7867-7874.