کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10532613 | 961634 | 2013 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABAA receptors
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
![عکس صفحه اول مقاله: Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABAA receptors Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABAA receptors](/preview/png/10532613.png)
چکیده انگلیسی
This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the Ï1 and α1 subunits of GABAA receptors, for localizing reagents of interest to the target receptor. A cysteine residue was inserted at the C terminus of the cysteine-lacking B-domain (Bd) and used for conjugating maleimide-containing compounds. As determined by enzyme-linked immunosorbent assay (ELISA), binding of a Bd-cys-S-fluorescein conjugate to polyclonal guinea pig anti-GABAA-Ï1 and rabbit anti-GABAA-α1 IgG was similar to that exhibited by full-length protein A. Surface plasmon resonance analysis of the interaction of Bd-cys-S-PEG3400-biotin conjugate (where PEG is polyethylene glycol) with anti-GABAA-Ï1 and anti-GABAA-α1 yielded KD values of 6.4 ± 1.9 and 0.4 ± 0.1 nM, respectively. Fluorescence anisotropy analysis of the binding of Bd-cys-S-fluorescein to the two antibodies yielded EC50 values of 65 and 18 nM, respectively. As determined with biotin-reactive fluorescent reagents, Bd-cys-S-PEG3400-biotin specifically bound to the plasma membrane of Xenopus laevis oocytes that expressed α1β2γ2 or homomeric Ï1 GABAA receptors and were pretreated with the corresponding anti-GABAA IgG. The IgG-binding specificity and high affinity of Bd-cys conjugates illustrate the potential of these conjugates, in combination with a selected IgG, to localize compounds of interest at specific cell surface proteins.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 432, Issue 1, 1 January 2013, Pages 49-57
Journal: Analytical Biochemistry - Volume 432, Issue 1, 1 January 2013, Pages 49-57
نویسندگان
Nasser M. Qtaishat, Hélène A. Gussin, David R. Pepperberg,