Selected article for: "SDS PAGE loading buffer and western blotting"
Author: Cobucci-Ponzano, Beatrice; Conte, Fiorella; Benelli, Dario; Londei, Paola; Flagiello, Angela; Monti, Maria; Pucci, Piero; Rossi, Mosè; Moracci, Marco
Title: The gene of an archaeal a-l-fucosidase is expressed by translational frameshifting
  • Document date: 2006_8_18
    • ID: 69gftii4_12
    Snippet: For the western blot studies, equal amounts of E.coli cultures expressing the wild-type and mutant fucA1 genes, normalized for the OD 600 , were resuspended in SDS-PAGE loading buffer containing 0.03 M Tris-HCl buffer, pH 6.8, 3% SDS (w/v), 6.7% glycerol (w/v), 6.7% 2-mercaptoethanol (w/v) and 0.002% blue bromophenol (w/v). The samples were incubated at 100 C for 5 min (unless otherwise indicated) and were directly loaded on to the gel. Western b.....
    Document: For the western blot studies, equal amounts of E.coli cultures expressing the wild-type and mutant fucA1 genes, normalized for the OD 600 , were resuspended in SDS-PAGE loading buffer containing 0.03 M Tris-HCl buffer, pH 6.8, 3% SDS (w/v), 6.7% glycerol (w/v), 6.7% 2-mercaptoethanol (w/v) and 0.002% blue bromophenol (w/v). The samples were incubated at 100 C for 5 min (unless otherwise indicated) and were directly loaded on to the gel. Western blot analyses were performed by blotting SDS-PAGEs of the concentrations indicated on Hybond-P polyvinylidenfluorid filters (Amersham Biosciences, Uppsala, Sweden); polyclonal anti-Ssa-fuc antibodies from rabbit (PRIMM, Milan, Italy) and anti-GST antibodies (Amersham Biosciences) were diluted 1:5000 and 1:40 000, respectively. The filters were washed and incubated with the ImmunoPure anti-rabbit IgG antibody conjugated with the horseradish peroxidase (HRP) from Pierce Biotechnology (Rockford, IL, USA). Filters were developed with the ECL-plus Western Blotting Detection system (Amersham Biosciences) by following the manufacturer's indications. The molecular weight markers used in the western blot analyses were the ECL streptavidin-HRP conjugate (Amersham Biosciences).

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