Optimization of western blotting for the detection of proteins of different molecular weight
Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended...
| Publié dans: | BioTechniques. - 1991. - 68(2020), 6 vom: 05. Juni, Seite 318-324 |
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| Auteur principal: | |
| Autres auteurs: | , , , |
| Format: | Article en ligne |
| Langue: | English |
| Publié: |
2020
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| Accès à la collection: | BioTechniques |
| Sujets: | Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S. CFTR LAMP1 Rab11a Towbin’s transfer buffer blocking agents methanol concentration optimization plus... |
| Résumé: | Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0-20%) in Towbin's transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins |
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| Description: | Date Completed 31.08.2021 Date Revised 11.11.2023 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/btn-2019-0124 |