Download Nucleic Acid Aptamers: Selection, Characterization, and by Günter Mayer PDF
By Günter Mayer
This quantity presents protocol references protecting contemporary advancements within the aptamer box. in the final decade, aptamers became an increasing number of renowned, and their subtle biophysical houses including their skill to be simply converted and, hence, tailored to numerous regimens makes them a really promising classification of compounds. Divided into 3 sections, the publication covers choice, a sequence of analytical ways to check biophysical homes of aptamer-target interactions, in addition to a number of purposes of aptamers. Written for the hugely winning Methods in Molecular Biology sequence, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and pointers on troubleshooting and heading off recognized pitfalls.
Practical and simple to keep on with, Nucleic Acid Aptamers: choice, Characterization, and Application offers a state of the art precis of contemporary advancements within the aptamer box and should be a invaluable source for scientists within the lifestyles sciences operating with aptamers as instruments to clarify organic systems.
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Additional resources for Nucleic Acid Aptamers: Selection, Characterization, and Application
Sample text
Data points represent mean values of ten independent measurements (further details see [11]) SELEX of Cell-Specific RNA Aptamers 29 Fig. 2 (a) Flow cytometry analysis. Upon binding of Atto-647 N-labeled Aptamer AIR-3A (red) to BAF/gp130/IL6R/TNF cells presenting the human IL-6 receptor, fluorescence shifts to higher values. Cells incubated with the variant G17U (Atto647N-labeled control, black) resulted in a smaller shift compared to AIR-3A. Laser scanning microscopic image of AIR-3A (b) or its variant G17U (c) with BAF/gp130/IL6R/TNF cells.
5 Binding Assay 1. 5. 2. Enzyme for dephosphorylation: phosphatase alkaline (PA) 1 U/μl. 3. Inactivation of PA: 200 mM ethylene glycol tetraacetic acid (EGTA). 4. 2, 50 mM DTT, 1 mM EDTA, 1 mM spermidine. 5. Enzyme for phosphorylation: T4 Polynucleotide Kinase 10 U/μl. 6. Incubation buffer for treatment: culture medium serum-free. 7. Washing buffer: culture medium serum-free. Developing Aptamers by Cell-Based SELEX 39 8. 6 % sodium dodecyl sulfate (SDS). 9. [γ-32P]ATP (6000 Ci/mmol). 10. Nonspecific competitor: polyinosinic acid (Poly-I).
Intensity of signals was measured for Axl and other two members (Dtk and Mer) belonging to the same receptor family. Pixel intensity is reported in the graph (b). A clear reduction of Axl phosphorylation was observed. Figure partially reproduced from Ref. T aptamer by phospho-RTK array analysis is shown in Fig. 5 (adapted from Ref. [23]). T aptamer shows to inhibit Axl phosphorylation. This circumstance has been further validated in our laboratory by different approaches [23]. 4 Notes 1. Dissolve urea by adding it slowly.