Co-immunoprecipitation assay (Co-IP)

Description

Co-immunoprecipitation is the most commonly used method to test whether two proteins of interest are associated in vivo. In our group, we developed co-immunoprecipitation techniques to study protein-protein interactions among WSSV ORFs and the host proteins. Briefly, two proteins of interest are cloned into V5-tagged (pDHsp/V5-His) and FLAG-tagged (pDHsp/FLAG-His) expression plasmids, respectively, and co-transfected into Sf9 insect cells. If the two proteins interact, then complexes consisting of V5-tagged protein plus FLAG-tagged protein will be formed. There complexes are coimmunoprecipitated by anti-FLAG antiserum and detected by Western blotting using anti-V5 antibody.

Brief protocol

Sf9 cells were seeded on six-well plates (8 ´10⁵ cells/well) and cotransfected with 2 μg of V5-tagged pDHsp/V5-His and 2 μg of FLAG-tagged pDHsp/FLAG-His expression plasmid, using Cellfectin reagent. After transfection for 16 to 18 h, the cells were heat shocked in a 42°C water bath for 30 min and then returned to 27°C. Six hours after being heat shocked, the cells were washed with PBS and lysed in 100 μl of NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) supplemented with a protease inhibitor cocktail tablet. The lysis procedure was carried out on ice for 10 min with occasional shaking. The lysate was centrifuged at 12,000 ´g for 5 min, and an aliquot of the supernatant (10 μl) was reserved for immunoblot analysis to confirm the expression of the transfected gene. The remaining supernatant (90 μl) was then incubated with 15 μl of anti-FLAG M2 affinity gel (Sigma) at 4°C overnight with rotation. The gel was then washed five times in 150 μl of NP-40 lysis buffer. Aliquots of the total cell lysates and immunoprecipitates were separated by 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. V5-tagged fusion proteins were detected with rabbit anti-V5 antibody (Sigma) and goat anti-rabbit immunoglobulin G–horseradish peroxidase conjugate (Sigma). FLAG-tagged fusion protein was detected with mouse anti-FLAG monoclonal antibody (Sigma) and goat anti-mouse immunoglobulin G–horseradish peroxidase conjugate (Sigma).

Some of our publications that have used this platform

1. Chang, Y. S. Liu, W. J., Chou, T. L., Lee, Y. T., Lee, T. L., Huang, W. T., Kou, G, H. and Lo, C. F. (2008) Characterization of White Spot Syndrome Virus Envelope Protein VP51A and its Interaction with Viral Tegument Protein VP26. J Virol 82: 12555-12564.
2. Liu, W. J., Chang, Y. S., Wang, H. C., Leu, J., H. Kou, G, H. and Lo, C. F. (2008) Transactivation, Dimerization, and DNA-binding Activity of White Spot Syndrome Virus immediate early protein IE1. J Virol 82: 11362-11373.
3. Leu, J. H., Wang, H.C., Kou, G.H., Lo, C.F. (2008) Penaeus monodon caspase is targeted by a white spot syndrome virus anti-apoptosis protein. Dev Comp Immunol 32: 476-486.
4. Leu, J.H., Kuo, Y.C., Kou, G.H., Lo, C.F. (2008) Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon. Dev Comp Immunol 32: 121-133.