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Protein spots were excised from stained 2D gels, destained
, digested with trypsin (sequence grade, Pro-mega, Madison, USA) at 37 [degrees]C overnight (6ng/[mu]l).
The gel was then dried for 5 min, stained in acid violet for 5 min, destained
, and finally dried.
After the gels were dried at 65[degrees]C for 10 min, they were stained with 5 g/L amido schwarz in a 50 mL/L acetic acid solution for 4 min and destained
before a final drying at 75[degrees]C for 8 min.
The sample lanes were destained
in methanol, rinsed in Tris-buffered saline with Tween 20 (TBST) buffer (containing 10 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, and 0.5 mL/L Tween 20), and incubated for 30 min with 1:10 diluted cow colostrum in TBST buffer.
Gels were destained
in 50 mL/L acetic acid for at least 2 h.
Nations who do not rise are destained
to dust bin of history.
After incubation, gel was stained with coomassie brilliant blue R250 for 30 min and then destained
in 5% methanol, 5% glacial acetic acid.
The gels were destained
with 40% v/v ethanol and 10% v/v acetic acid until visualization of spots.
Paraffin slices were dewaxed and rehydrated, then stained with hematoxylin (Beyotime Biotechnology, China) for 3 min, washed for 5 min, destained
with hydrochloric acid alcohol, washed for 5 min, and fast stained with eosin (Beyotime Biotechnology, China).
The resolved protein was either stained using Coomassie blue reagent overnight and destained
with 50% dd[H.sub.2]O, 40% methanol and 10% acetic acid before imaging or transferred to a nitrocellulose membrane using the TransBlot-Semidry Transfer System (Bio-rad) for the Western blotting.