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)
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.
2. Centrifuge at 13,000 rpm for 10 sec at 4°C in a microcentrifuge.
3. Transfer the supernatant (precleared cell lysate) to a fresh tube.
4. Set up two tubes containing equal amounts of the precleared cell lysate.
i. Add 50 µl of glutathione-Sepharose beads (50/50 slurry in lysis buffer) to each tube.
ii. Then add GST protein to one tube and the GST fusion probe to the other (~5-10 µg each).
The amount of protein added should be equimolar in the two reactions (i.e., the final molar concentration of GST should be the same as that of the GST probe protein).
5. Incubate the tubes for 2 h at 4°C with end-over-end mixing.
6. Centrifuge the samples at 13,000 rpm for 10 sec at 4°C in a microcentrifuge.
7. Transfer the supernatants to fresh microcentrifuge tubes and reserve them for SDS-PAGE (see Troubleshooting).
8. Wash the beads four times with 1 mL of ice-cold lysis buffer. Discard the washes.
9. At this point, proteins bound to the probe protein must be dissociated for analysis. Use either the boiling method (the more popular choice; see Step 10) or one of the elution methods described in Step 11.
The disadvantage of elution (Step 11) is that if multiple elutions are necessary, the final sample volume may be large. In these cases, it may be impossible to load more than a small percentage of the sample on an SDS-polyacrylamide gel for analysis. This is the reason most researchers choose instead to boil complexes off the beads in SDS sample buffer (Step 10).
10. If the samples are to be boiled off the beads, do as follows:
i. Add an equal volume of 2X SDS gel-loading buffer to the beads.
It is important to include a "glutathione-Sepharose beads only" control. Proteins bound nonspecifically to the beads can appear as bound to the fusion protein, even in comparison to GST alone.
ii. Boil for 5 min in a water bath.
Samples are now ready for SDS-PAGE (Step 12).
11. If the samples are to be eluted from the beads, perform one of the following options:
Option I:
i. Add 50 µl of 20 mM reduced glutathione in 50 mM Tris-Cl (pH 8.0).
ii. Flick the tube to mix the contents, and incubate the samples at room temperature for 5 min.
iii. Centrifuge the samples at 13,000 rpm for 10 sec at room temperature in a microcentrifuge.
iv. Transfer the supernatant containing the eluted protein to a new microcentrifuge tube.
v. If elution is incomplete, repeat Steps 11.i-11.iv.
Option II:
vi. Add 50 µl of 20 mM reduced glutathione in 50 mM Tris-Cl (pH 8.0).
vii. Flick the tube to mix the contents, and incubate the samples at room temperature for 5 min.
viii. Transfer the mixture to a fresh 0.5-mL tube. Carefully, using a sterile, small-bore needle, poke a hole in the bottom of the 0.5-mL tube. Place it inside a 1.5-mL microcentrifuge tube.
ix. Centrifuge the tubes together at 1000g in a microcentrifuge for 2 min at room temperature.
The eluted proteins will be deposited in the 1.5-mL tube.
12. Analyze as much of the sample as possible by SDS-PAGE (see SDS-Polyacrylamide Gel Electrophoresis of Proteins).
13. Detect the proteins. The method of detection will depend on the experimental goal:
i. If the goal is to detect the 35S-labeled proteins associated with the fusion protein after SDS-PAGE, dry the gel on a gel dryer and expose it to X-ray film.
ii. If the goal is to detect specific partners after SDS-PAGE, transfer the proteins to a membrane and subject them to Western analysis.
iii. If the goal is to determine the size and abundance of proteins associated with the fusion protein from a nonradioactive lysate, subsequent to SDS-PAGE, stain the gel with Coomassie blue or silver stain.
TROUBLESHOOTING
Problem: Conditions for pull-down are not optimal
[Step 13]
Solution: Analyze aliquots of equal percentage volumes (e.g., 1%) from each of the following fractions generated during the protocol:
Total cell lysate (Step 1)
Beads prior to elution (Step 9)
Eluate (Steps 10 or 11)
Beads post-elution (Steps 10 or 11)
Supernatant saved at Step 7
"Beads + GST" eluate (if applicable; Steps 10 or 11)
"Beads alone" eluate (if applicable; Steps 10 or 11)
After these samples are collected at the indicated steps, add an appropriate volume of SDS-PAGE sample buffer. Snap-freeze the samples in a dry-ice ethanol bath for analysis by SDS-PAGE (during Step 12), and perform autoradiography, Western analysis, or Coomassie staining as appropriate (Step 13).
Results from this gel will show:
The prevalence of the novel interactor in the total cell lysate (aliquot from Step 1).
How much of the interactor is bound to the GST fusion protein (aliquot from Step 9).
How much GST fusion protein + associated proteins were eluted from the beads (aliquot of eluate from Steps 10 or 11).
What fraction of the interactors remained bound (beads remaining from Steps 10 or 11).
How much of the interacting protein was depleted from the total cell lysate (aliquot from supernatant at Step 7).
Problem: Low signal
[Step 13]
Solution: If an interaction yields a low signal even though the interactor is abundant in the total cell lysate, this may indicate that the binding conditions are not optimal. A change in salt and detergent concentrations, in addition to increasing the time allowed for association, may improve the binding. A poor signal can also be caused by inefficient elution; i.e., the complex being retained on the glutathione-Sepharose beads. This can be determined by SDS-PAGE comparison of the eluate versus the "beads post-elution" fraction (Steps 10 or 11). If this proves to be the problem, it may be remedied by pooling multiple elutions or increasing the time for elution. Releasing the interactors by boiling in sample buffer (Step 10), if appropriate, would be applicable in this case.
Problem: Nonspecific background
[Step 13]
Solution: Preclearing a lysate with GST, or beads alone, can help to minimize nonspecific interactions (see Step 1). Titrating the amount of lysate added and increasing the stringency of the binding and wash conditions can also reduce background.
DISCUSSION
When analyzing an interaction by Western blot (e.g., analyzing a predicted interaction for which there are available antibodies), it is important to reprobe the membrane with anti-GST antibodies after probing for the candidate interactor. This will determine whether all samples were incubated with the same amount of GST fusion protein, and it will help to determine whether the fusion protein is undergoing degradation while incubated with the cell lysate. If the interactors are radio- or biotin-labeled, one should also confirm equal amounts of GST fusion and GST proteins by Western blot or Coomassie blue staining.
REFERENCES
Dignam, J.D., Lebowitz, R.M., and Roeder, R.G. 1982. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11: 1475–1489.
Orlinick, J.R. and Chao, M.V. 1996. Interactions of the cellular polypeptides with the cytoplasmic domain of the mouse Fas antigen. J. Biol. Chem. 271: 8627–8632.[Abstract/Free Full Text]
Spector, D.L., Goldman, R.D., and Leinwand, L.A. 1998. Cells: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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