Fluorescence Staining
Reagents needed:
methanol
acetic acid
ruthenium II tris (bathophenanthroline disulfonate) (RuBPS)
Procedure:
1. Fix gels in 20% methanol, 10% acetic acid solution with two changes of fixing solution (total of 3 hr to overnight).
2. Stain gel in 20% methanol, 10% acetic acid and 2.4 mM RuBPS for 4 hr to overnight.
3. Wash gel twice with 10% acetic acid for 1 hr each.
4. Wash gel with 5% acetic acid.
5. Imaging is performed using a Molecular Imager FX Pro Plus (BioRad).
Notes:
1. The excitation wavelengths of RuBPS are 300 nm and 480 nm and emission is 618 nm.
2. Use plastic trays; dye will bind to glass.
3. Rabilloud et al. state that acetic acid quenches fluorescence. We find that the acetic acid is essential.
Reference
Rabilloud T. Strub JM. Luche S. van Dorsselaer A. Lunardi J. (2001) A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 1(5):699-704.
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Protocols
The Michigan Proteome Consortium follows a standard set of protocols listed below:
Whole cell lysate
Membrane proteins
IPG 1st dimension
SDS page
Colloidal coomassie stain
Fluorescence stain
Silver stain
In-gel digestion
References and suggested reading
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