On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate.
We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.
Iwafune
Yuko
Y
Yokohama City University, International Graduate School of Arts and Sciences, Suchiro 1-7-29, Tsurumi, Yokohama 230-0045, Japan.
Tan
Jian-Zhong
JZ
Ino
Yoko
Y
Okayama
Akiko
A
Ishigaki
Yuji
Y
Saito
Koji
K
Suzuki
Nobutake
N
Arima
Mikiko
M
Oba
Mitsuyoshi
M
Kamei
Shuichi
S
Tanga
Michifumi
M
Okada
Takeshi
T
Hirano
Hisashi
H
D000818:Animals; D002244:Carbon / Q000737:chemistry; D002417:Cattle; D018130:Diamond / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry; D040081:Protein Array Analysis / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D013193:Stainless Steel / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D013499:Surface Properties
D006023:Glycoproteins; D011506:Proteins; D013388:Succinimides; D013193:Stainless Steel; D002244:Carbon; D018130:Diamond; C001426:N-hydroxysuccinimide