Rapid and easy protein staining for SDS-PAGE using an intramolecular charge transfer-based fluorescent reagent.
High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.
Suzuki
Yoshio
Y
Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology, AIST, c/o Katayanagi Advanced Research Laboratories, Tokyo University of Technology, Tokyo, Japan.
Yokoyama
Kenji
K
Namatame
Ichiji
I
D000818:Animals; D001923:Brain Chemistry; D002919:Chymotrypsinogen / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007074:Immunoglobulin G / Q000032:analysis; D051379:Mice; D009281:Naphthalenes / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000032:analysis; D009570:Nitriles / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods*; D014168:Transferrin / Q000032:analysis
C514698:2-(2-(2-(4-hydroxynaphthalen-1-yl)vinyl)-6-methylpyran-4-ylidene)malononitrile; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D009281:Naphthalenes; D009419:Nerve Tissue Proteins; D009570:Nitriles; D011506:Proteins; D014168:Transferrin; D012710:Serum Albumin, Bovine; D002919:Chymotrypsinogen