Cart  |  Log In

Focus: Protein:Protein Interactions

Non-Radioactive Identification of Protein:Protein Interactions Using TNT® SP6 High-Yield Protein Expression System

The TNT® SP6 High-Yield Protein Expression System (Cat.# L3261) and FluoroTect™ GreenLys in vitro Translation Labeling 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.

By Trista Schagat, Ph.D., Natalie Betz, Ph.D. and Jacqui Mendez, M.S.
Promega Corporation

Published in March 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 Mix30µl
DNA Template
pF3 WG Flexi Vector (c-fos)3µg
or
pF3 WG Flexi Vector (c-jun)3µg
or
Luciferase SP6 Control DNA8µg
FluoroTect™ GreenLys tRNA4µl
Nuclease-Free Water to a final volume50µl

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.

thumbnail-Non-radiographic detection of protein expression and immunoprecipitations.
Non-radiographic detection of protein expression and immunoprecipitations.

Figure 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.

Reference

  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, 23–27.