Development of a Method for Evaluating Protein Tyrosine Phosphatase CD45 Inhibitors Using Jurkat Cell Membrane
✍ Scribed by Deena Waddleton; Chidambaram Ramachandran; Qingping Wang
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 89 KB
- Volume
- 285
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A simple, high-throughput fluorescent assay was developed to measure the inhibition of membrane-bound CD45 from Jurkat cells. This assay is based on the fact that ϳ64% of PTP activity from Jurkat cell membrane is contributed by CD45. This has been proven by comparing the activity in membrane protein from wildtype Jurkat cells and CD45-negative mutant cells, and also by measuring the residual activity after depleting CD45 from Jurkat cell membrane. We have demonstrated that fluorescein diphosphate can be used as a substrate to monitor CD45 activity from Jurkat cell membrane, which allows us to easily follow CD45 activity in both fluorescent and absorbance modes in a 96-well format. Some common protein tyrosine phosphatase inhibitors have been evaluated with this assay.