High-throughput negative detection of SDS-PAGE separated proteins and its application for proteomics.
A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, low-cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.
Cong
Wei-Tao
WT
School of Pharmacy, Wenzhou Medical College, Zhejiang, PR China.
Hwang
Sun-Young
SY
Jin
Li-Tai
LT
He
Hong-Zhang
HZ
Choi
Jung-Kap
JK
D004396:Coloring Agents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004801:Eosine Yellowish-(YS) / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D007093:Imidazoles / Q000737:chemistry; D016014:Linear Models; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015032:Zinc / Q000737:chemistry
D004396:Coloring Agents; D029968:Escherichia coli Proteins; D007093:Imidazoles; D011506:Proteins; C029899:imidazole; D015032:Zinc; D004801:Eosine Yellowish-(YS)