GST pull-down Protocol

GST-PULL DOWN手册 冷泉港 protocol——GST pull-down Protocol GST Pull-down Margret B. Einarson, Elena N. Pugacheva, and Jason R. Orlinick This protocol was adapted from "Identification of Protein-Protein Interactions with Glutathione-S-Transferase Fusion Proteins," Chapter 6, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION Glutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. One of these applications is their use as probes for the identification of protein-protein interactions. The pull-down method described in this protocol is fundamentally similar to immunoprecipitation. Immunoprecipitation is based on the ability of an antibody to bind to its antigen in solution, and the subsequent purification of the immunocomplex by collection on protein A- or G-coupled beads. Similarly, the GST pull-down is an affinity capture of one or more proteins (either defined or unknown) in solution by its interaction with the GST fusion probe protein and subsequent isolation of the complex by collection of the interacting proteins through the binding of GST to glutathione-coupled beads.

RELATED INFORMATION An introduction to the use of GST fusion proteins for studying protein-protein interactions can be found in Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins. This protocol is designed to use a 35S-labeled cell lysate as the source for interacting proteins. For 35S-labeling procedures, see Orlinick and Chao (1996) and Spector et al. (1998). Additionally, if the interacting protein of interest is known to be confined to a specific cellular compartment (e.g., the nucleus), a fraction of the cell lysate corresponding to that compartment (e.g., a nuclear extract [to prepare, see Dignam et al. 1982]) can be used in place of a total cell lysate.

MATERIALS This procedure may require equipment or reagents for Western analysis, Coomassie blue staining, and/or silver staining (see Step 13). Reagents Cell lysate (unlabeled or labeled with 35S, depending on experimental goal) This experiment compares GST versus GST fusion protein, so it is necessary to prepare enough lysate to provide equal amounts of lysate in each reaction. The amount of lysate needed to detect an interaction is highly variable. Start with lysate equivalent to 1 x 106 to 1 x 107 tissue culture cells. GST fusion protein (see Preparation of GST Fusion Proteins) GST protein GST pull-down lysis buffer, ice cold Reagents for SDS-Polyacrylamide Gel Electrophoresis of Proteins (see Step 12) 2X SDS gel-loading buffer Tris-Cl (50 mM, pH 8.0) containing 20 mM reduced glutathione (optional; for Step 11 only) GST-PULL DOWN手册 Equipment Equipment for SDS-Polyacrylamide Gel Electrophoresis of Proteins (see Step 12) Gel dryer (optional; see Step 13) Glutathione-Sepharose beads (store at 4°C; do not freeze) Beads are often supplied by commercial vendors in solutions containing alcohols. It is important to wash the beads thoroughly in GST pull-down lysis buffer and to generate a 50/50 slurry of beads in GST pull-down lysis buffer prior to use. Microcentrifuge, precooled to 4°C Microcentrifuge tubes, 0.5 mL (optional; for Steps 11.vi-11.ix only) and 1.5 mL Needle, small bore, sterile (optional; for Steps 11.vi-11.ix only) Rotator for end-over-end mixing Water bath, boiling (optional; see Step 10) X-ray film (optional; see Step 13)

METHOD 1. Incubate the cell lysate with 50 μL of glutathione-Sepharose beads (50/50 slurry in lysis buffer) and 25 μg of GST (NOT the GST fusion probe protein) for 2 h at 4°C with end-over-end mixing. Allow enough volume in the tube to permit liberal mixing; 500 μl to 1 mL is a good starting point. This step is designed to preclear from the lysate proteins that interact nonspecifically with the GST moiety or with the beads alone. If the interaction will be detected primarily with antibodies directed to a candidate interacting protein, it is not absolutely necessary to preclear the lysates with GST or glutathione-Sepharose beads. However, when 35S-labeled cell lysates are used to identify novel protein-protein interactions, these steps can help to reduce background. When detecting the interacting protein with antibodies to that protein, it is important to include "GST + beads" and "beads alone" controls.