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2.1K Views
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08:00 min
November 4th, 2021
DOI :
10.3791/62971-v
Chapters
0:04
Introduction
0:44
Peptide Extraction: Cell Culture
1:30
Peptide Extraction: Animal Tissues
3:39
Peptide Quantification with Fluorescamine
4:18
Reductive Methylation of Amines Labeling
5:58
Results: Determining Labelled Peptides Using Mass Spectroscopy
7:19
Conclusion
Transcript
在这里,我们描述了一种基于蛋白酶快速热灭活的教学方法,以保留天然细胞内肽,避免细胞和组织中的降解,然后使用同位素标记对肽进行相对定量。这种标记方法具有很多优点。这些试剂是市售的,价格低廉,化学稳定,并且可以在单个血清中分析多达五个样品。
首先在含有15%FBS和1%青霉素链霉素的DMEM中培养人神经母细胞瘤细胞。将细胞保持在37摄氏度,低于5%的二氧化碳。用PBS洗涤细胞两次,然后加入10毫升PBS,刮擦细胞,并将它们收集到15毫升的管中。
以800倍G离心细胞五分
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Summary
本文介绍了一种基于热灭活的样品制备方法,以保存内源性肽,避免死后降解,然后使用同位素标记和LC-MS进行相对定量。
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