SYPRO

Blotting stains SYPRO Ruby (Blot) 2-8 3 50 min N.C. and PVDF1... 

Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P., BioTechniques, 12 564 (1992). j SYPRO is a trademark of Molecular Probes, Inc., Eugene, OR. k SYPRO Ruby IEF stain requires an overnight incubation. 

IPGs into mirrors. An easy way to stain IPGs is to immerse them for 1 h in colloidal CBB G-250 followed by two 10-min water washes. There is also a version of SYPRO Ruby stain specifically formulated for use with both carrier ampholyte and IPG-IEF gels. 

CBB G-250 is another popular total protein stain. Researchers blotting 2-D PAGE gels particularly favor it because it is compatible with mass spectrometry. Stained blots provide good media for archiving 2-D PAGE separations. A version of SYPRO Ruby, formulated for blots, is a very sensitive total protein stain. 

Figure 5.1 Two-dimensional electrophoresis separates potato proteins based on their isoelectric points and molecular weights. Using a wide-scale pH gradient and large gels, 1000-2000 quantifiable proteins can be separated. The gel stained with SYPRO Rubi shows soluble tuber proteins of potato cultivar Sante.

Yan, J. X. Harry, R. A. Spibey, C. Dunn, M. J. Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 2000, 27(17), 3657—3665. 

Fluorescent dyes have also been recently introduced for membrane staining. For example, SYPRO Ruby protein blot stain is a new,... 

White IR, Pickford R, Wood J, Skehel JM, Gangadharan B, Cutler P. A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis. Electrophoresis 2004 25(17) 3048-3054. 

Ideally, the gel should have been stained with a Coomassie stain—preferably a colloidal Coomassie if detection sensitivity is an issue, e.g., (2,3), or one of the commercially available stains. This method is also compatible with Sypro stains (though an ultraviolet transilluminator will be required to visualize the protein bands or spots during excision). Standard silver stains are not compatible, but certain modified silver stains—e.g., (4), or one of the commercially available mass spectrometry compatible silver stains—can be used however, even these usually give inferior mass spectrometry data compared to Coomassie- or Sypro-stained gels. 

Figure 13 Molecular-mass analysis of phosphorylase B (panel B) and the separation of the noncovalently (Sypro Red) labeled protein markers (panel A ALA, a-lactalbumin CBA, carbonic anhydrase OVA, ovalbumin BSA, bovine serum albumin BGA, (f-galactosidase) and the covalently (FITC) labeled protein markers (panel C TRI, tripsin inhibitor CAH, carbonic anhydrase ADH, alcohol dehydrogenase BSA, bovine serum albumin BGA, P-galactosidase). Separation conditions gel, 1% agarose, 2% linear polyacrylamide (LPA, MW 700,000-1,000,000) in 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation buffer, 50 mM Tris, 50 mM TAPS, 0.05% SDS (pH 8.4) separation voltage, 420 V, current, 5 mA gel thickness, 190 pm effective separation length, 3.5 cm temperature, 25°C sample loading, 0.2 pL into 2.5 x 4 x 0.19-mm injection wells. Sample buffer contained 0.05% SDS and 1 x Sypro Red. (Reproduced with permission from Ref. 141.)...

Rabilloud, T, Strub, J.M., 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, 699-704. 

Zubkov, M. V., Fuchs, B. M., Eilers, H., BurkhiU, P. H., and Amann, R. (1999). Determination of total protein content of bacterial cells by SYPRO staining and flow cytometry. Appl. Environ. Microbiol. 65(7), 3251-3257. 

This is an entirely gel-based procedure no mass spectrometry is involved. Proteins from the two comparison samples are separated by a 2-DE protocol and images of the stained spots are captured after staining the gels with a suitable dye (e.g., CCB or SYPRO Ruby) next, the measured densities of the spots are compared using image analysis software to provide differential expression of proteins.44 45 Identification of proteins is accomplished as mentioned in Section 9.12.3. 

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