Abstract 216: Tracking expression, post-translational modifications and interactions of EGF signalling proteins in A431 cells with antibody microarrays
Notice bibliographique
Résumé
Abstract Elucidation of epidermal growth factor (EGF) signalling pathways in cancer cells has helped to define the mechanisms for their neoplastic transformation and potential drug targets for therapeutic intervention. Antibody microarrays are promising tools to evaluate alterations in the levels and phosphorylation status of hundreds of proteins of interest with only microgram amounts of crude cell and tissue lysate protein. However, interpretations of the results from traditional antibody microarray approaches have been hampered by the problems associated with sample preparation and protein detection, even when reliable antibodies are deployed in these arrays. The Kinex™ KAM-900P antibody microarray permitted semi-quantitative measurements of the expressions, post-translational modifications and interactions of proteins with 100 µg or less of lysate proteins. These microarrays utilize approximately 878 different pan- and phosphosite-specific antibodies for tracking protein kinases, phosphatases and other low abundance regulatory proteins. Multiple detection protocols were developed with the KAM-900P slides to enable high depth profiling of protein levels, phosphorylation and protein-protein interactions in A431 cells in response to EGF treatment. One method (KAM) involved the capture of in vitro biotin-labeled proteins, followed by their detection with a secondary dye-labeled anti-biotin antibody. False positive signals from associated proteins in complexes with the targets were reduced by chemical cleavage with NTCB prior to their capture on the array, and this also produced more uniformity of the dye signals for protein targets despite vast differences in their sizes. Transient changes in protein phosphorylation in EGF treated cells that were typically lost when processed by conventional methods were better preserved by chemical cleavage right at time of sample homogenization. Biotin-labeling and subsequent detection of the protein on the array with a dye-labeled secondary antibody further reduced non-specific background signals, allowed for a greater dynamic range of detection, and enhanced discrimination of subtle changes. In conjunction with other detection protocols, such as the usage of dye-labeled reporter antibodies for generic protein-tyrosine phosphorylation in sandwich antibody microarrays (SAM format) or generic protein phosphorylation with nanoparticles such as pAMIGO (PAM format), it is also feasible to monitor changes in general post-translational modifications of target proteins or their specific association with other adapter, scaffolding and chaperone proteins of interest for which antibody probes are available. Citation Format: Lambert Yue, Steven Pelech. Tracking expression, post-translational modifications and interactions of EGF signalling proteins in A431 cells with antibody microarrays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 216. doi:10.1158/1538-7445.AM2017-216
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».