Visualising intraparticle protein transport in porous adsorbents by confocal microscopy

被引:98
作者
Ljunglöf, A
Thömmes, J
机构
[1] Univ Dusseldorf, Inst Enzymtechnol, D-52426 Julich, Germany
[2] Amersham Pharm Biotech, S-75182 Uppsala, Sweden
关键词
adsorption; confocal scanning microscopy; proteins; finite bath adsorption; protein diffusion;
D O I
10.1016/S0021-9673(98)00378-1
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Confocal scanning microscopy was used to study protein uptake to porous adsorbents during batch experiments in a finite bath. By coupling of a fluorescent dye to the protein molecules the penetration of single adsorbent particles at different times during batch uptake could be observed visually. Intensity profiles of the protein distribution within a single particle were obtained by horizontal scanning. After integration of the profiles the overall fluorescence within a bead could be calculated. Relating the overall fluorescence at different incubation times to the value at equilibrium allowed the construction of the fractional approach to equilibrium versus time. These data were compared to uptake curves, which had been obtained by measurements of the protein concentration in the supernatant and an excellent agreement of the curves was detected The procedure was performed for two different proteins (lysozyme and human IgG) on two different media for protein adsorption (SP Sepharose Fast Flow and SP Sepharose XL; Pharmacia Biotech) and in all cases it could be shown, that the results from the direct measurements by confocal microscopy correspond very well to the data obtained from the indirect measurements in the fluid phase. (C) 1998 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:387 / 395
页数:9
相关论文
共 8 条
[1]   MODELING AND ANALYSIS OF BIOSPECIFIC ADSORPTION IN A FINITE BATH [J].
ARVE, BH ;
LIAPIS, AI .
AICHE JOURNAL, 1987, 33 (02) :179-193
[2]   3-DIMENSIONAL MICROSCOPY USING A CONFOCAL LASER SCANNING MICROSCOPE [J].
CARLSSON, K ;
DANIELSSON, PE ;
LENZ, R ;
LILJEBORG, A ;
MAJLOF, L ;
ASLUND, N .
OPTICS LETTERS, 1985, 10 (02) :53-55
[3]   CONFOCAL IMAGING FOR 3-D DIGITAL MICROSCOPY [J].
CARLSSON, K ;
ASLUND, N .
APPLIED OPTICS, 1987, 26 (16) :3232-3238
[4]  
HORSTMANN BJ, 1989, CHEM ENG RES DES, V67, P243
[5]   In situ measurements of ion-exchange processes in single polymer particles: Laser trapping microspectroscopy and confocal fluorescence microspectroscopy [J].
Kim, HB ;
Hayashi, M ;
Nakatani, K ;
Kitamura, N ;
Sasaki, K ;
Hotta, JI ;
Masuhara, H .
ANALYTICAL CHEMISTRY, 1996, 68 (03) :409-414
[6]   Simulation of light attenuation within fluorescent microspheres used for liquid fraction separation recorded by a CSLM [J].
Liljeborg, A .
THREE-DIMENSIONAL MICROSCOPY: IMAGE ACQUISITION AND PROCESSING III, 1996, 2655 :11-17
[7]   Confocal microscopy as a tool for studying protein adsorption to chromatographic matrices [J].
Ljunglof, A ;
Hjorth, R .
JOURNAL OF CHROMATOGRAPHY A, 1996, 743 (01) :75-83
[8]  
PAWLEY JB, 1995, HDB BIOL CONFOCAL MI