Wide-Ranged Fluorescent Molecular Weight Size Markers for Electrophoresis

Abstract

A molecular weight size marker (MWSM) is commonly used in gel electrophoresis for protein and nucleic acid analyses. In electrophoresis, biomolecules are detected by a wide variety of labels such as direct tagging, antibody, and silver stain, often with a set of protein or DNA standards for molecular weight based visual identification of analytes. Development of enhanced MWSMs has been made to ease the size indications, and visible and fluorescent rainbow MWSMs have been well-acknowledged for the convenience of color-based size indications to overcome limitations and difficulties in use of identically stained markers (GE healthcare and Invitrogen, US). However, increasing use of fluorescent molecules from a wide range of emission (em) wavelengths (400 to 700 nm) needs MWSM with a broader fluorescence spectrum. Currently only a few numbers of MWSMs with limited wavelengths are known and commercially available to date. For example, GE Healthcare’s latest MWSM of dual indication with visible and fluorescent dyes is useful for proteomics analysis, but the use of Cy3 and Cy5 dyes limits it detection range. Here in, we report our effort to develop a wide ranged fluorescent MWSM, which covers upto near-infrared wavelengths, consisted of sequentially arranged cyanine dyes enabling proteomic analysis without post-stain for size indication. The development of new MWSM system that can cover all range of the fluorescent wavelength is an imperative task. Previously reported cyanine dyes equipped with vinyl sulfone linker (FPG-456, FPR-553 and FPR-648) were utilized in this work due to their optimal stability and robustness for protein conjugation. The absorption (abs) wavelength of FPG-456 is 495 nm and its em wavelength is 522 nm, and those of FPR-553 and FPR-648 are 551 nm/570 nm and 648 nm/672 nm (abs/em), respectively. Combination of three dyes can create a range of emission spectra from 450 to 700 nm. Commonly used six standard proteins, phosphorylase b (97.6 kDa), albumin (66 kDa), glyceraldehyde-3-phophate dehydrogenase (35 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and lysozyme (14 kDa) were used as size indicating markers (Aldrich, US). The conjugation between a protein and a dye was precisely followed previously reported protocol. In brief, 1.0 eq of given protein and 1.0 eq of vinyl sulfone dye were incubated in aqueous pH 8.0 phosphate or carbonate buffer (0.1 M) at 36 oC for 4 h to afford corresponding protein-dye complex which was stored and used in the analyses without purification. From the product solution, the dye-conjugated protein solution (1 μL) was diluted to 200 folds in PBS. Optical properties of products were measured with a fluorescence plate reader with a monochromator (EnSpire, Perkin-Elmer, Germany). Figure 1(a) shows abs spectra of six FPG-456 dye stained protein complexes (left), comparison of both abs (dot, middle) and em intensities (bar, middle) and SDS-PAGE bands (right) under irradiation of maximum abs wavelength. Figures 1(b) and 1(c) are those of FPR-553