Tris acetate sds page

Peptides from the N-terminus of Dop (ISTSTPQKNDEHQEQC and MSRQEGAASRPADGAC) were synthesised and used to immunise rabbits and to affinity purify antibodies (anti-Dop 1303) (Eurogentec). Protein extracts from staged embryos were prepared in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% β-mercaptoethanol) and run on 3-8% Tris acetate SDS-PAGE (Invitrogen) and transferred to PVDF membrane (Whatman, GE Healthcare) for immunoblotting. For 2D PAGE, protein extracts from visually staged embryos were acetone precipitated and redissolved in rehydration buffer [7 M urea, 2 M thiourea, 1.2% CHAPS, 0.25% ampholytes (GE Healthcare), 0.4% ASB-14, 43 mM DTT]. Protein concentration was established using 2-D Quant (GE Healthcare). Equal amounts of protein were separated on pH 4-7 Immobiline DryStrip gels (Amersham) with the IPGphor isoelectric focusing system (Amersham). The second dimension was performed on 4-12% bis-Tris Zoom gels (Invitrogen). Signal intensity on immunoblots was measured using ImageJ.
For dephosphorylation assays, 100 μg protein lysate was incubated with 5 µl λ-protein phosphatase (New England BioLabs) at 30°C for 30 min. As control, phosphatase was heat inactivated for 1 hour at 65°C in the presence of 50 mM EDTA.

67NR and 4T1 cells were a gift from L. Wakefield (NCI, Bethesda [16] (link)). Protein was extracted by cell lysis on ice for 30 minutes in Golden Lysis buffer (10 mM Tris pH 8.0+400 mM NaCl +1% Triton X-100+10% Glycerol+Complete protease inhibitor cocktail (Roche)+phosphatase inhibitor (Sigma)). 30 µg (or as indicated) total protein extract in NuPage LDS Sample Buffer (Invitrogen) and 0.25% beta-mercaptoethanol were resolved on 3%–8% Tris-Acetate SDS-PAGE (NuPage, Invitrogen), transferred onto PVDF membrane (Millipore), blocked in 5% skim milk and incubated overnight with the following primary antibodies at 4°C: mouse anti E-cadherin (BD) 1∶5000, mouse anti beta-catenin (BD) 1∶1000, mouse anti vimentin (Sigma) 1∶5000, and mouse anti beta-actin (Abcam 1∶10000. Membranes were then incubated with horse-radish peroxidase linked anti-mouse (GE Healthcare) and immunoblots visualized using Amersham ECL Prime Western Blotting Detection System and Amersham Hyperfilm ECL (GE Healthcare).