Responsibility edited by Julie D. Physical description xvii, p. Series Methods in molecular biology Clifton, N. Online Available online. SpringerLink Full view. SAL3 off-campus storage. M45 V.
More options. Find it at other libraries via WorldCat Limited preview. Contributor Thompson, Julie D. Schaeffer-Reiss, Christine. Ueffing, Marius. Bibliography Includes bibliographical references and index.
Functional Proteomics Techniques to Isolate and Characterize the Human Proteasome
Exosomes Joost P. Hegmans, Peter J. Gerber, and Bart N. Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure. Email or Customer ID. Forgot password? Old Password. New Password. Password Changed Successfully Your password has been changed. Returning user. Request Username Can't sign in? Forgot your username? Enter your email address below and we will send you your username. Three constructs were purchased from the Kazusa Institute, each containing one of the human proteasome subunits. E, see TM for details. The cells were harvested 48 hours after transfection.
Methods and Protocols
The media was removed, and the cells were washed with 25ml of cold PBS. The cells were scraped from the surface and collected into conical tubes. G, G The gel was run at V for 1. After mixing at rpm for 30 seconds on a plate shaker, the samples were incubated at room temperature for 30 minutes.
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Mass spectrometry was performed by MS Bioworks www. Gel pieces were processed using a robot ProGest, DigiLab. Data was processed by MS Bioworks using Mascot. Mascot DAT files were parsed into the Scaffold software for validation, filtering and to create a non-redundant list for each sample. Labeling was performed at room temperature for 15 minutes. The reactions were stored on ice. Figure 3.
Figure 4. Labels under the figure specify the protein used for the pull-down. Before sending samples for mass spectrometry analysis, eluates were assayed for proteolytic activity. Each substrate detects a different protease activity. Substrate cleavage generates a luminescent signal produced by the luciferase reaction, with the signal proportional to the amount of proteasome activity. All three proteasome subunit pull-down samples contained all three protease activities. Activity measurements ranged from 7- to fold above background.
These samples resulted in no significant activity, indicating the activity from the proteasome pull-down eluates are from isolated proteasome and not background proteins or ProTEV Plus data not shown. Figure 5.www.dogsandtrail.com/images/jijetiru/2747-juegos-de-las.php
Functional Proteomics - Xing Wang, Matthew Kuruc - Bok () | Bokus
Following cleavage by the 20S proteasome, the substrate for luciferase aminoluciferin is released, resulting in light production by the luciferase reaction. The samples were analyzed as described in the methods section. The mass spectrometric analysis detected 31 of the 34 proteasome subunits Figure 6.
The three missing subunits were small 20S core proteins and might have been missed because of size or inaccessibility. The results show a high percentage enrichment of proteasome subunits. Figure 6. To demonstrate the advantages of using cell-free translation systems in protein mapping experiments, we mapped the interactions between PSMD2, 4 and 8. Figure 7, Panel A, diagrams the experimental procedure.
These studies map direct interactions between these proteins that is consistent with studies published for the yeast proteasome REF.
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Figure 7. Using cell-Free expression to map proteasome interactions. Protein:protein interaction scheme. The labels above the gels indicate the bait proteins, and the labels below the gel indicate prey proteins. Red lettering represents no interaction, and green lettering represents positive interaction.
The diagram below the gel represents the interaction map between these proteasome subunits.
We used commercial products and services from Promega Corporation, the Kazusa Institute and MS Bioworks to quickly isolate subunits of the human proteosome, test their activity and characterized the subunit interactions. Finally, cell-free protein expression was used as an easy and fast method for confirming and mapping protein:protein interactions discovered by mass spectrometry.
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