Western blot analysis of sub-cellular fractionated samples using the Odyssey Infrared Imaging System
Source Yukiko Misawa et al (University of Virginia)
Introduction
This protocol describes nuclear and cytoplasmic fractionation of tissue culture cells and a method for Western blot detection of proteins using the Odyssey Infrared Imaging System. This protocol was used to detect expression of the "small" Tap protein in 293T, HeLa and COS cells. The Odyssey system has several advantages over the more widely used chemiluminescent detection methods: 1) both Alexa Fluor 680 and IRDye800 are stable and the membranes can be stored before scanning 2) in general this system allows better quantification 3) the two-color detection system makes it possible to simultaneously detect two different proteins using secondary antibodies from different species (see Figure 1) 4) electronic data files allow the intensity of bands to be varied as needed.
Materials
Reagents
•Triton X-100 lysis buffer: 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 137.5 mM NaCl, 10% Glycerol, 1 mM sodium vanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 5 mM EDTA, and Protease Inhibitor Cocktail (SIGMA; P2714)
•2xSDS-PAGE sample buffer: SDS-PAGE sample buffer; 125 mM Tris-HCl (pH 6.8), 4% SDS, 2% 2-mercaptoethanol, 20% glycerol, and 0.3% BTB
•Pre-stained blue protein size maker: Precision Plus Protein Standards (Bio-Rad)
•PVDF membrane: Immobilon-FL membrane (Millipore)
•Transfer buffer: 3.33 g Trizma base and 14.4 g Glycine /1L H2O with 10% MeOH, prepare 10X buffer without MeOH.
•Primary antibody
•Secondary antibodies
1) anti-rabbit IgG: Alexa Fluor 680 goat anti-rabbit IgG (Molecular probes, Invitrogen, http://probes.invitrogen.com, #A21076)
use 1:50,000 dilution
2) anti-mouse IgG: IRDye800 conjugated anti-mouse IgG (Rockland Immunochemicals, http://www.rockland-inc.com/, #610-132-121)
use 1:20,000 dilution
3) anti-goat IgG: Alexa Fluor 680 rabbit anti-goat IgG (Molecular Probes, Invitrogen, http://probes.invitrogen.com, #A21088)
use 1: 20,000 dilution