The Use of 2-D Gels to Identify Novel Protein–Protein Interactions in the Cochlea

Abstract

Functional proteomics comprises a wide range of technologies for the identification of novel protein-protein interactions and biological markers. Studies of protein-protein interactions have gained from the development of techniques and technologies such as immunoprecipitation, preparative two-dimensional (2-D) gel electrophoresis for peptide mass fingerprinting (PMF), using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These applications enabled the discovery of putative protein partners without a priori knowledge of which one(s) might be relevant. Here, we report the methods by which membrane proteins are isolated from cochlear tissues and prepared for identification by mass spectrometry techniques.

The large-conductance Ca2+-activated K+ channel interacts with the apolipoprotein ApoA1

“…Eight cochleae from 3 week-old chickens, including sensory epithelium, ganglion and supporting cells were removed and processed as described previously [14, 15]. The sample was centrifuged and the pellet solubilized in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 5 mM EDTA, 50 mM NaF, 500 μg/ml AEBSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μM okadaic acid, and 0.1% ASB-14 (Calbiochem), followed by vortexing and agitating on a rocker for 1 h at 4°C.…”

A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea

“…Coimmunoprecipitation-A total of 16 cochleae were excised from 30-day-old CBA/J mice and immersed in 100 l of lysis buffer containing 50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 5 mM EDTA, 50 mM NaF, 500 g/ml AEBSF, 10 g/ml leupeptin, 10 g/ml pepstatin A, 2 g/ml aprotinin, and 5 M okadaic acid, as described previously (16) and sonicated (Sonic Dismembrator Model 100; Thermo Fisher). The resulting lysate was centrifuged for 2 min at 700 ϫ g and the supernatant removed to another tube.…”

Exploiting Protein Interaction Networks to Unravel Complex Biological Questions

“…This step is practical, especially for high-throughput experiments involving mass spectrometry (Kathiresan et al, 2009;). After clearing debris, nuclei, etc., separate the membrane fraction from other soluble proteins using ultracentrifugation, by spinning the sample at 100k x g for about an hour at 4 o C. The pellet will contain membrane from the plasmalemma, mitochondrion, and endoplasmic reticulum, while the supernatant will contain any remaining soluble proteins.…”

“…At this point there are various nuances in terms of technique for running a 2-D gel. Among these is a step-by-step description by Kathiresan et al, (2009). Here, we will suggest some of the initial troubleshooting that may be necessary before and/or after running a full-fledged experiment.…”

“…These descriptions will include coimmunoprecipitation, verification using reciprocal coimmunoprecipitation and RNAi, and data mining of specific databases. Their purpose is to provide a primer without going into specific details, since many have been described previously in great detail (e.g., Golemis & Adams, 2005;Kathiresan et al, 2009;Navaratnam, 2009). …”