Staining Sypro

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

As with the gel stain, fluorescent colors have also caused a small revolution in blot staining. SYPRO Ruby stains proteins on PVDF or nitrocellulose with almost the same sensitivity as gold—certainly with more sensitivity than Coomassie and India ink. Staining takes just about three quarters of an hour bathe nitrocellulose membranes for 2 x 10 minutes in 7% acetic acid and 10% methanol, and then stain for 15 minutes with SYPRO Ruby and finally wash with water for 6 x 1 minutes. 

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. 

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. 

This green fluorescent Pro-Q Emerald 300 glycoprotein-staining method can he combined with SYPRO Ruby for total protein staining, with sensitivity up to... 

Sypro Ruby Protein Blot stain (Cat. No. S-11791 Invitrogen/Molecular Probes). 

The limited volume of the total cellular material from biopsy samples makes it necessary to use a slight modification of the lysis buffer recipe as compared with the cell culture lysis buffer. The following protocol describes a method for preparing whole cell lysates from LCM cells. The limited biopsy sample generally precludes the use of total protein assays before microarray printing. To compensate for this, one microarray slide is stained for total protein using a Sypro Ruby Protein blot stain (see Subheading 3.3.3.). 

Each microarray is probed with a single primary antibody directed against the protein of interest. Microarray slides used for immunostaining should be blocked before staining. Microarray slides used for Sypro Ruby protein staining do not require blocking. 

Sypro Ruby Total Protein Staining of Microarray... 

Sypro Ruby blot stain is a ruthenium complex, exhibiting luminescence upon excitation with either UV-B or blue light. The luminescence may be visualized with UV epi-illumination sources, UV or blue-light transilluminators, or laserscanning instmments with excitation light at 450, 473, 488, or 532 nm. The emission peak of the ruthenium complex is approximately 618 nm (24). 

Detection methods based on the post-electrophoretic staining of proteins with fluorescent compounds have the potential of increased sensitivity combined with an extended dynamic range for improved quantitation. The most commonly used reagents are the SYPRO series of dyes from Molecular Probes (Patton, 2000). Extensive studies have been carried out to evaluate the sensitivity of these fluorescent dyes compared with silver staining (Berggren et al., 2002 Lopez et al., 2000). In our laboratory (Yan et al.,... 

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