Avidin functional affinity electrophoresis (AFAEP) is substituted for an avidin affinity column (AAC) to capture biotinylated peptides in the Isotope‐Coded Affinity Tagging (ICAT) technique which is a valuable tool in quantitative proteomics. In this new technique, the AFAEP‐captured ICAT‐labeled biotinylated peptides are extracted with the biotin tag intact from the polyacrylamide gel piece with aqueous 95% formamide (pH 8.2) at 65 °C for 20 min, and then detected by a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometer. Bovine serum albumin (BSA) and the 12C‐ and 13C‐ICAT reagents are used to test this AFAEP‐ICAT technique. The results show that both AFAEP and AAC methods provide quantitative information of the relative amounts of 12C‐ and 13C‐ICAT‐labeled biotinylated tryptic peptides of BSA in a sample. Compared with AAC, the AFAEP is cheaper to perform, more stringent in capturing the biotinylated peptides, and capable of simultaneously processing multiple samples. 相似文献
Horseradish peroxidase (HRP) is immobilized in three easy steps on SiO(2) surfaces with the help of a polycationic second generation dendronized polymer (denpol) and the biotin-avidin system. This stepwise immobilization process is monitored and quantitatively analyzed with the transmission interferometric adsorption sensor. Partially biotinylated denpol is first adsorbed onto SiO(2) , followed by addition of avidin and then of biotinylated HRP. Denpols in their molecular structure combine properties of polymers as well as dendrimers which are found to be of clear advantage for this type of non-covalent enzyme immobilization. With respect to the reproducibility of the adsorption process and with respect to the stability of the adsorbed polymer layer, the denpol is superior to α-poly-D-lysine which is used as a reference polymer. Furthermore, HRP immobilized with the denpol on commercial glass slides remains considerably more active upon storage as compared to HRP immobilized with the help of α-poly-D-lysine with a similar number of repeating units. The ease of the denpol-mediated HRP immobilization and the high stability of the immobilized enzyme are promising for bioanalytical applications. 相似文献
New methods are needed to modify silk biomaterials with bioactive molecules for tissue engineering and drug delivery. In the present study, silk fibroin in solution or in microsphere format was coupled with NeutrAvidin via carbodiimide chemistry. Silk fibroin retained its self‐assembly features after reaction. It was found that more than four NeutrAvidin molecules bound to one silk molecule. Non‐specific binding of biotin or NeutrAvidin to silk microspheres could be reduced by pre‐treatment of the microspheres with BSA or post‐treatment with detergent. The NeutrAvidin‐coupled silk microspheres were coupled with biotinylated anti‐CD3 antibody and the functionalized microspheres were able to specifically bind to the CD3 positive T‐lymphocytic cell line Jurkat.
The kinetics of free glucose-6-phosphate dehydrogenase (G-6-PDH), biotinylated G-6-PDH, and biotinylated G-6-PDH complexed
with avidin were investigated. The kinetics of the free enzyme were consistent with a sequential rather than a ping-pong mechanism.
The kinetics of the biotinylated enzyme were similar to that of the free enzyme, but the kinetic constants were different;
theKm value for NADP was halved, whereas theKm for G-6-P decreased only slightly. In the presence of avidin, theKm of biotinylated G-6-PDH for G-6-P nearly doubled whereas theKm for NADP did not change significantly. Avidin complexed with biotinylated G-6-PDH inhibited the enzyme from acting. Based
upon these reactions, it was possible to devise assays for either free biotin or free avidin using biotinylated G-6-PDH as
the indicator enzyme. Concentrations of biotin between 40 and 60 mg/mL, or of 25–95 Μg/mL of avidin could be measured within
2 min through the use of biotinylated G-6-PDH. 相似文献
Cell adhesion to a scaffold is a prerequisite for tissue engineering. Many studies have been focused on enhancing cell adhesion to synthetic materials that are used for scaffold fabrication. In this study, we applied an avidin-biotin binding system to enhance chondrocyte adhesion to biodegradable polymers. Biotin molecules were conjugated to the cell membrane of chondrocytes, and mediated cell adhesion to avidin-coated surfaces. We demonstrated that immobilization of biotin molecules to chondrocyte surfaces enhanced cell adhesion to avidin-coated biodegradable polymers such as poly(L-lactic acid), poly(D,L-lactic acid), and polycaprolactone, compared to the adhesion of normal chondrocytes to the same type of biodegradable polymer. The biotinylated chondrocytes still maintained their proliferation ability. This study showed the promise of applying the avidin-biotin system in cartilage tissue engineering. [diagram in text]. 相似文献
The multilayer thin films composed of polymers, avidin, and concanavalin A (Con A) were prepared on the surface of a quartz slide by alternate deposition of anionic and cationic polymers and the proteins. The loading of avidin and Con A in the film was evaluated spectroscopically by using Texas Red tagged avidin and Con A. Avidin molecules assembled in the film caused its binding activity toward biotin and analogue 2-(4′-hydroxyphenylazo)benzoic acid to be retained. The polymer layers contributed to improving stability of the protein film.
Polymer/Con A/avidin composite film on a solid surface. 相似文献