Academic Journals Database
Disseminating quality controlled scientific knowledge

Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes

ADD TO MY LIST
 
Author(s): Eric B. Haura | Roberto Sacco | Jiannong Li | André C. Müller | Florian Grebien | Giulio Superti-Furga | Keiryn L. Bennett

Journal: Journal of Integrated OMICS
ISSN 2182-0287

Volume: 2;
Issue: 1;
Start page: 55;
Date: 2012;
Original page

Keywords: EGFR | Grb2 | Orbitrap | TAP | downscale

ABSTRACT
In this study we show that via stable, retroviral-expression of tagged EGFR del (L747-S752 deletion mutant) in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a Flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR del complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material.
Why do you need a reservation system?      Save time & money - Smart Internet Solutions