Isolation and Digestion of Neutrophil Proteins

Abstract

Source:
Tia Rizakos¹, Jennifer Geddes-McAlister<sup>1

1</sup>Molecular and Cellular Biology Department, University of Guelph.

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 

1. 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. 
2. Discard the supernatant, and add 15 mL of phosphate-buffered solution (PBS) into the tube.
3. Centrifuge samples at 400 x g for 5 min.
4. Repeat steps 1.2 to 1.3. 
   NOTE: Cell samples can be flash frozen with ....