Practical aspects of fluorescent staining for proteomic applications

SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-f ormat-two dimensional (2-D) gels, are compared to the manufacturer's recomm ended protocols to determine sensitivity and reproducibility of the new met hods. This study examines the critical aspects of fixation, washing, and st aining to develop an optimized fluorescent staining method. It was determin ed that careful control of sodium dodecyl sulfate (SDS) levels and pH in th e gel was critical for successful staining with SYPRO Orange. Overnight fix ation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental e ffects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, fo llowed by staining with SYPRO Orange diluted 1:5000 with washing solution f or 3 or more hours, produced high sensitivity, tow background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no s ignificant changes with respect to the initial protein patterns, and allowe d successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of ST ORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Rub y is also discussed.