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Protein detection onto PVDF membranes

2-D PAGE and electroblotting onto PVDF membranes have become widely used techniques for the characterization of proteins. Recent improvements have allowed a higher protein load, a better transfer yield and a higher sensitivity in automated protein microsequencing. However, the application of these techniques to proteins would not have been possible without the development of complementary detection methods. Amido Black, Coomassie Brilliant Blue R-250, colloidal gold and Ponceau S are commonly utilized to visualize proteins on PVDF membranes and are compatible with the ensuing Edman degradation chemistry [3].

Amido Black

After electrotransfer, the PVDF membranes were stained in a solution containing Amido Black (0.5% w/v), isopropanol (25% v/v) and acetic acid (10% v/v) for 2 min. Destaining was done by several soakings in deionized water [3].

Coomassie Brilliant Blue R-250

After electrotransfer, the PVDF membranes were stained in a solution containing Coomassie Brilliant Blue R-250 (0.1% w/v) and methanol (50% v/v) for 15 min. Destaining was done in a solution containing methanol (40% v/v) and acetic acid (10% v/v) [3].

Colloidal Gold (Progold)

After electrotransfer, the PVDF membranes were incubated in PBS-Tween 0.5% for 30 minutes, washed 3 x 5 min. in PBS-Tween 0.5% and 1 min. in deionized water. They were stained in 100 ml of Problot solution overnight.

Ponceau S

After electrotransfer, the PVDF membranes were stained in a solution containing Ponceau S (0.2% w/v) and TCA (3% v/v). Destaining was done by several soakings in deionized water [3].

Drying

The PVDF stained membranes were either air dried or dried on a 3 mm thick plate onto an heating plate at 37o C for 10 min [3].

Scanning

The Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel) from Molecular Dynamics and the GS-700 from Bio-Rad have been used as scanning device. This scanners were linked to Sparc workstations and Macintosh computers.



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