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Non-Radioactive Identification of Protein:Protein Interactions

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Abstract

The TnT® SP6 High-Yield Protein Expression System (Cat.# L5001) were used to express non-radioactively labeled c-fos and c-jun proteins. Reactions expressing the two proteins were then co-incubated, and protein:protein interactions were detected by co-immunoprecipitation with an anti-c-fos antibody.

Trista Schagat, Natalie Betz and Jacqui Mendez

Promega Corporation
Publication Date: 2007

Introduction

Identification of protein:protein interactions is critical to understanding protein function. The high yields of the TnT® SP6 High-Yield Protein Expression System make it an attractive system for cell-free expression of proteins for subsequent protein:protein interaction studies. This article describes a fast protocol for detecting c-fos and c-jun interaction by co-immunoprecipitation directly from TnT® reactions without the need for prior protein purification or for the addition of affinity tags.

Materials and Methods

For optimal expression in the TnT® SP6 High-Yield Protein Expression System, the open reading frame of c-fos (Accession# K00650) and c-jun (Accession# J04111) were cloned into the pF3A WG (BYDV) Flexi® Vector (Cat.# L5671). Three separate TnT® reactions were assembled following the standard protocol(1) .

TnT® SP6 High-Yield Master Mix 30µl
DNA Template (choose one of the three below)
pF3 WG Flexi Vector (c-fos) 3µg
or
pF3 WG Flexi Vector (c-jun) 3µg
or
Luciferase SP6 Control DNA 8µg
FluoroTect™ GreenLys tRNA 4µl
Nuclease-Free Water to a final volume 50µl

Reactions were incubated at 25°C for 2 hours. Prior to loading on a gel, reactions were treated with RNase ONE™ Ribonuclease to remove unincorporated, fluorescent, charged tRNA (1 unit/ml, 5 minutes at 37°C)(2) .

Immunoprecipitations were carried out using the following protocol. All incubations were at room temperature with constant rotation to ensure adequate mixing.

  1. Either 10µl each of the c-fos and c-jun TnT® reactions or 10µl each of the luciferase and c-jun TnT® reactions were mixed with 50µl Immunoprecipitation Buffer (20mM Tris (pH 7.5), 150mM NaCl and 0.2% Triton® X-100) for a final volume of 70µl and incubated for 1 hour.
  2. To each immunoprecipitation, 0.6µg anti-fos rabbit polyclonal antibody (Santa Cruz cat.# sc-52) was added and incubated for 1 hour.
  3. To each immunoprecipitation, 5µl protein A agarose (Sigma cat.# P7786) was added and incubated for 1 hour.
  4. Immunoprecipitates were collected by centrifugation at 7,000 × g for 10 seconds. The supernatant was carefully removed, and immunoprecipitates were washed by resuspending in 0.5ml Immunoprecipitation Buffer. This was repeated for a total of 4 washes.
  5. The final pellet was suspended in 20µl 1X LDS Sample Buffer, heated at 70°C for 3 minutes, centrifuged briefly, and 20µl was loaded onto a gel.

TnT® reactions and immunoprecipitation supernatants were run on 4–12% Novex NuPAGE® Bis-Tris gels in MES running buffer using LDS sample buffer (Invitrogen). Fluorescent protein was detected using a Hitachi FMBIO® II instrument set on the 505nm channel.

Results and Conclusions

The results of the expression and co-immunoprecipitation of c-fos and c-jun are shown in Figure 1. The successful expression of c-fos (50kDa), c-jun (39kDa) and the control luciferase (62kDa) protein are shown in lanes 2, 3 and 4. Expression was easily detected using a fluorescent scanner. The co-immunoprecipitation of c-jun by c-fos and the anti-c-fos antibody is clearly visible in lane 5. When luciferase was substituted for c-fos in the immunoprecipitation, no significant amount of c-jun was precipitated. This shows that the precipitation was specific to the interaction between c-fos and c-jun.

6423TAFigure 1. Non-radiographic detection of protein expression and immunoprecipitations.

TNT® SP6 High-Yield reactions were performed with FluoroTect™ GreenLys in vitro Translation Labeling System. Lanes 1–4 are 5µl of TNT® reactions expressing each of the following: lane 1, no template control; lane 2, c-fos; lane 3, c-jun; lane 4, luciferase control. Immunoprecipitations using the anti-c-fos antibody were performed directly from mixtures of the TNT® reactions: lane 5, c-fos and c-jun immunoprecipitation; lane 6, luciferase and c-jun immunoprecipitation.

These data show that the c-fos:c-jun interaction can be detected directly in the TnT® reactions without the need for prior protein purification or preclearing of the extracts with protein A agarose. The TnT® SP6 High-Yield Protein Expression System yielded sufficient protein in a single reaction for multiple immunoprecipitation experiments. Use of the FluoroTect™ GreenLys in vitro Translation Labeling System allowed for non-radiographic labeling and fast in-gel detection of proteins. Together, these systems gave the means for rapid in vitro screening of protein:protein interactions in a single-day procedure.

References

  1. TnT® SP6 High-Yield Protein Expression System Technical Manual, TM282, Promega Corporation.
  2. Kobs, G. et al. (2001) FluoroTect™ GreenLys in vitro Translation Labeling System. Promega Notes 77, 23–7.

How to Cite This Article

Schagat, T., Betz, N. and Mendez, J. Non-Radioactive Identification of Protein:Protein Interactions Using TnT® SP6 High-Yield Protein Expression System. [Internet] 2007. [cited: year, month, date]. Available from: http://kr.promega.com/resources/pubhub/enotes/nonradioactive-protein-protein-interaction-identification/

Schagat, T., Betz, N. and Mendez, J. Non-Radioactive Identification of Protein:Protein Interactions Using TnT® SP6 High-Yield Protein Expression System. Promega Corporation Web site. http://kr.promega.com/resources/pubhub/enotes/nonradioactive-protein-protein-interaction-identification/ Updated 2007. Accessed Month Day, Year.

Products may be covered by pending or issued patents or may have certain limitations on use. Please visit our patent and trademark web page for more information.

FMBIO is a registered trademark of Hitachi Software Engineering Company, Ltd. NuPAGE is a registered trademark of Invitrogen Corporation. Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology Corporation.

Figures

6423TAFigure 1. Non-radiographic detection of protein expression and immunoprecipitations.

TNT® SP6 High-Yield reactions were performed with FluoroTect™ GreenLys in vitro Translation Labeling System. Lanes 1–4 are 5µl of TNT® reactions expressing each of the following: lane 1, no template control; lane 2, c-fos; lane 3, c-jun; lane 4, luciferase control. Immunoprecipitations using the anti-c-fos antibody were performed directly from mixtures of the TNT® reactions: lane 5, c-fos and c-jun immunoprecipitation; lane 6, luciferase and c-jun immunoprecipitation.

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