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Isolation and Digestion of Neutrophil Proteins
Overview
This video demonstrates the protein isolation from neutrophils, which is further processed and digested into smaller peptides. These peptides can be used for future analysis.
Protocol
1. Neutrophil Cell Collection and Lysis
- Collect neutrophil-differentiated HL60 cells in a 15 mL tube by centrifuging samples at 400 x g for 5 mins.
NOTE: Neutrophils should be at a confluency of 3.5 x 105 cells/mL after differentiation. Harvest approximately five million cells for total proteome extraction. - Discard the supernatant, and add 15 mL of phosphate-buffered solution (PBS) into the tube.
- Centrifug.......
Representative Results
Figure 1. Workflow of HL60 cells differentiated into neutrophils for mass spectrometry-based proteomics profiling. Undifferentiated and differentiated (via DMSO chemical stimulation) HL60 cell lines were cultured, collected, and processed for total proteome extraction. Proteins were digested with Trypsin/LysC, and peptides were purified using C18 STAGE-tips, followed by detection by LC-MS/MS. Max.......
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