Data Item _em_sample_preparation.details


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Details of the specimen preparation
    Detergent-solubilized particles eluted from the cation-exchange
    column were directly adsorbed for 1 min to parlodion carbon-coated
    copper grids rendered hydrophilic by glow discharge at low pressure
    in air. Grids were washed with 4 drops of double-distilled water
    and stained with 2 drops of 0.75% uranyl formate.
  Selectively stained by injection of horseradish peroxidase,
  embedded in Spurr's resin and cut into 2-3 um thick sections.
  S. cerevisiae PDC was purified to near homogeneity
  from baker's yeast by modification of a published procedure. Highly
  purified E1 was obtained by resolution of PDC with 2 M NaCl at pH 7.3
  followed by FPLC on a Superdex 200 column. The weight-average molecular
  weight of the PDC was determined by light scattering measurement to be
  ~8 x 106. On the basis of the known molecular weight of the complex and
  its component enzymes and the experimentally determined polypeptide chain
  ratios of E2/BP/E3, we estimated that the subunit composition of the
  S. cerevisiae PDC is ~24 E1 tetramers, 60 E2 monomers, 12 BP monomers,
  and 8 E3 dimers. Sufficient E1 was added to a sample of the PDC
  preparation to increase the molar ratio of E1/E2 core to 60:1.
   embedded in vitreous ice.

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