Removal of nonspecific binding proteins is required in co-immunoprecipitation with nuclear proteins
Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the d...
| Publié dans: | BioTechniques. - 1993. - 73(2022), 6 vom: 01. Dez., Seite 289-296 |
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| Auteur principal: | |
| Autres auteurs: | , , , , , , , |
| Format: | Article en ligne |
| Langue: | English |
| Publié: |
2022
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| Accès à la collection: | BioTechniques |
| Sujets: | Journal Article Research Support, Non-U.S. Gov't NP-40 Western blot agarose beads co-immunoprecipitation cytoplasmic and nuclear protein extraction nonspecific binding proteins nuclear proteins osmotic pressure plus... |
| Résumé: | Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the decrease of ion concentrations but reduced by Nonidet P40. Western blot indicated that p65 and β-actin were present as nonspecifically bound protein to the beads. p53 and β-actin were present in the CO-IP precipitates of nuclear proteins but pretreatment cleared the nonspecifically pulled down p53 and β-actin. These data suggest that magnetic beads are better for CO-IP, but preclearing is necessary to minimize false positive regardless of which bead is used, particularly for nuclear proteins |
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| Description: | Date Completed 19.12.2022 Date Revised 22.12.2022 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/btn-2022-0048 |