Determination of Protein S-Acylation State by Enhanced Acyl-Switch Methods
Autor: | Dionne Turnbull, Piers A. Hemsley, Charlotte H. Hurst |
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Rok vydání: | 2019 |
Předmět: |
0301 basic medicine
biology 010401 analytical chemistry N-Ethylmaleimide S-acylation 01 natural sciences Protein S 0104 chemical sciences Acylation 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Biochemistry Biotin chemistry biology.protein Protein precipitation lipids (amino acids peptides and proteins) Maleimide Cysteine |
Zdroj: | Methods in Molecular Biology ISBN: 9781493995318 |
Popis: | S-Acylation is increasingly being recognized as an important dynamic posttranslational modification of cysteine residues in proteins. Various approaches have been described for assaying protein S-acylation with acyl-switch approaches being the most common and accessible. However, these approaches can be time-consuming with low reproducibility as a result of multiple protein precipitation/resuspension cleanup steps. Here we present a faster, cleaner, and more sensitive acyl-switch approach for detecting the S-acylation state of any protein, from any cell or tissue type, that can be detected by western blotting. In the case of acyl-RAC, the procedure is now performed without protein precipitation, greatly increasing speed and improving sample handling in the assay. This also allows for more samples to be processed simultaneously and opens the way for medium-throughput assays. Overall, maleimide scavenging improves the reliability of determination and quantification of protein S-acylation state by acyl-switch methods. |
Databáze: | OpenAIRE |
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