Abstract
Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-d-biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.
Keywords: Avidin-immobilized sensor chip, HeLa, Membrane purification, MOLT-4, Plasma membrane, Surface plasmon resonance
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Glossary
- AEBSF
4-(2-aminoethyl)-benzenesulfonylfluoride
- BP
biotinylated protein(s)
- BSA
bovine serumalbumin
- DMSO
dimethyl sulfoxide
- E-64
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane
- EDC
N-ethyl-N¢-(3-diethylamino-propyl) carbodiimide
- GTA
3,3-dimethylglutaric acid
- HBS
HEPESbuffered saline
- HEPES
2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
- MEM
minimum essential medium
- NHS
N-hydroxysuccinimide
- OVA
ovalbumin
- PBS
phosphate-buffered saline
- RU
resonance unit
- SD
standard deviation
- SPR
surface plasmon resonance
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