التفاصيل البيبلوغرافية
| العنوان: |
High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics. |
| المؤلفون: |
Jeon, Won, Aceti, David, Bingman, Craig, Vojtik, Frank, Olson, Andrew, Ellefson, Jason, McCombs, Janet, Sreenath, Hassan, Blommel, Paul, Seder, Kory, Burns, Brendan, Geetha, Holalkere, Harms, Amy, Sabat, Grzegorz, Sussman, Michael, Fox, Brian, Phillips, George |
| المصدر: |
Journal of Structural & Functional Genomics; Sep2005, Vol. 6 Issue 2/3, p143-147, 5p |
| مستخلص: |
The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)6-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated ÄKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)6-MBP tags by TEV protease, (His)6-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and 15N and 13C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR. [ABSTRACT FROM AUTHOR] |
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| قاعدة البيانات: |
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