Western blotting

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Procedure

Proteins extracts (40µg) from striatum, motor cortex and cerebellum were denatured for 5 minutes at 95°C in loading buffer (1X) and separated on 4-12%-SDS polyacrylamide gels (Novex Pre-Cast gels, Invitrogene). The proteins were transferred to nitrocellulose membranes with a TransBlot cell apparatus (Bio-Rad). After blocking non-specific sites with 5% dried milk, membranes were incubated overnight at 4°C with primary antibody α-POL β (ab26343; 1:500, Abcam, Cambridge, UK), α-FEN1 (Abcam, Ab17993; 1:500), α-OGG1, (Abcam, Ab135940; 1:500), α-p53R2, (Abcam, Ab8105; 1:1000), APE1/Ref1 (sc5572; 1:100, Santa Cruz Biotechnology, USA), α-MUTYH (Abcam, Ab55551; 1:500) and for 1h at room temperature with α-β-Tubulin (Sigma, T5293; 1:2000) and α-LAMIN B1 (Abcam, ab16048; 1:10000). Immunoreactions were detected with appropriate α-rabbit or α-mouse peroxidase-conjugated secondary antibodies (Jackson immunoResearch Laboratories, 1:10000) and the ECL Western blotting detection Kit (Advansta). Signals were captured with ChemiDoc XRS system (Biorad) and quantified with Image Lab software.

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