Samples were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore). The membranes were then blocked with 5% skim milk in PBS with 0.05% Tween 20 (PBST) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies, anti-myc (1:5000, R950-25; Invitrogen) and anti-actin (1:600, Spring Bioscience) in 5% skim milk. After washing, the membranes were incubated for 1 h in horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technologies) at room temperature. The immunoreactive bands were visualised by enhanced chemiluminescence (ECL) and scanned using an LAS-3000 image analyser (FujiFilm).
Las 3000 image analyser
Manufactured by Fujifilm
Sourced in Japan
The LAS-3000 is an image analyser designed for laboratory use. It is capable of capturing and analysing digital images. The core function of the LAS-3000 is to provide users with tools for image acquisition and processing.
Automatically generated - may contain errors
2 protocols using las 3000 image analyser
1
Western Blot Protein Detection
2
Immunoblotting Analysis of Src and FAK Signaling
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Cell lysates and electrophoresis was performed as previously described [7 (link)]. Immunoblotting of Src (Y419; Millipore, USA) [14 (link)], Src kinase (Cell Signaling Technology, USA) [15 ], FAK (Y861; Invitrogen, UK) [16 (link)] and FAK (Y397; Abcam, UK) [17 (link)] were performed at 1:1000 dilutions with rabbit anti-human antibodies. Proteins were visualized by addition of a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit IgG. HRP-conjugated antibodies raised against beta tubulin were used as loading controls. HRP-conjugated protein was visualized using an enzyme-linked chemiluminescence reaction (Thermo Fisher Scientific, UK) and quantified using a LAS3000 image analyser (Fujifilm, Tokyo) and analysed with Image J software (NIH, US).
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