Protein interactions using far-Western blotting

Description

To identify proteins that interact with a given protein, far-western blotting is a useful platform with low experimental cost and high sensitivity. The principle of far-western blotting is modified from western blotting and uses the natively-folded recombinant protein as the first probe to interact with proteins that have been separated on a PVDF membrane. The second probe used in this platform is the specific antibody against the original protein, while theHRP-conjugated third probe is the antibody against the second probe. When combined with protein identification using LC-MS-MS, the resulting protein spots (or protein bands) can be characterized and then subjected to further functional assays, such as protein immuno-precipitation.

Brief protocol

1. Proteins were separated by 12% SDS-PAGE and then transferred to a nitrocellulose membrane. (We noted that 2D protein electrophoresis is also a good choice to separate protein mixture with good resolution.). Prior to the binding assay, the membrane was incubated with 5% skim milk in PBS buffer for 1 h. Following two washes, the membrane was equilibrated for 20 min with binding buffer (10 mM Tris-HCl [pH 6.5], 5 mM CaCl₂, 10 mM MgCl₂). Subsequently, the membrane was incubated with 0.8 mg of affinity-purified target protein (dialyzed against binding buffer at 4°C for 48 h) diluted in binding buffer with 0.02% skim milk and 1% Triton X-100. The membrane was extensively washed and reacted with antibody against target protein. After washing, the blot was incubated with secondary antibody-conjugated HRP (Roche). The membrane proteins that interacted with target protein were detected by the addition of HRP substrate (0.003% [wt/vol] 3',3'-diamino-benzenetetrahydrochloride [Sigma]-0.05% [wt/vol] H₂O₂).

References

1. Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW. (2006) PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol 80: 10734-10742.

Some of our publications that have used this platform

1. Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW. (2006) PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol 80: 10734-10742.
2. Wang HC, Wang HC, Ko TP, Lee YM, Leu JH, Ho CH, Huang WP, Lo CF, Wang AH. (2008) White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly. Proc Natl Acad Sci USA 105: 20758-20763.