ProteomIQ Blue, a Potent Post-stain for the Visualization and Subsequent Mass Spectrometry Based Identification of Fluorescent Stained Proteins on 2D-gels

Abstract

Manual spot excision for protein identification from fluorescent stained two-dimensional (2-D) gels is hard to accomplish. Here, we explore the use of ProteomIQ Blue as a post-stain method for the visualization of fluorescent stained/labeled proteins. We show that ProteomIQ Blue post-staining is almost as sensitive as staining with SYPRO Ruby or cyanine dyes alone. More than 90% of the protein spots that are stained with the fluorescent stains are still detectable with ProteomIQ Blue. In protein identification by mass spectrometry, ProteomIQ Blue post-stained spots provide high sensitivity and high protein sequence coverage of the peptide mass maps in both MALDI-TOF-MS and ESI-MS/MS analyses. In conclusion, post-staining of fluorescent stained gels with ProteomIQ Blue provides a facile and a powerful method to achieve quantitative protein analysis as well as protein identification in the same semianalytical gel without requiring sophisticated/expensive robotic equipment.

Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

“…Protein extracts were prepared as described by Wijte et al (Wijte et al 2006). In brief, cells were boiled for 10 min in a Tris-SDS solution (0.2% SDS, 0.028 M Tris-HCl, 0.022 Tris-base, and 0.2 M DTT) and subsequently a DNase/RNase treatment was given.…”

“…Proteomics analysis was performed using the 2D-DIGE method, according to Volkers et al (Volkers et al 2006) and Wijte et al (Wijte et al 2006). Protein samples were labeled according to the manufacturers’ protocol (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with CyDyes Cy3 and Cy5.…”

Polymethine cyanine dyes in β‐cyclodextrin solution: multiple equilibria and chemical oxidation

“…ICG was early recognized as a suitable NIR fluorogenic label, being used in several analytical and medical applications, including photodynamic therapy (PDT) 1, 2, 5–10. IR‐786 is widely used in enzymatic and immunoassays, DNA sequencing applications and it offers potential to target mitochondria due to its positive charge 1, 11–17…”

High Speed Isoelectric Focusing of Proteins Enabling Rapid Two-Dimensional Gel Electrophoresis

“…The strips were placed on the polyacrylamide gels without an agarose overlay and PAGE was performed immediately at 100V for 80 minutes. Gels were fixed a minimum of two hours in 25% ethanol, 10% acetic acid and stained overnight with colloidal Coomassie Brilliant Blue (CBB) G-250 (Wijte et al 2006, Smejkal 2004.…”