Isolation and Digestion of Neutrophil Proteins

Begin with a tube containing neutrophils. Add a buffer containing a proteinase inhibitor to prevent protein degradation.

Add a suitable detergent to permeabilize  the neutrophil membrane 

In an ice bath, sonicate the sample with high-frequency sound waves to disrupt the cell membrane, releasing intracellular contents, including various proteins.

Briefly centrifuge to collect contents from the tube walls and transfer them to a suitable tube.

Introduce a reducing agent and incubate at a high temperature to promote disulfide bond cleavage, causing the proteins to denature.

Cool and introduce an alkylating reagent that alkylates the sulfhydryl groups.

Add cold acetone to induce protein precipitation.

Centrifuge to pellet the proteins and cell fragments. Remove the supernatant, and air-dry.

Introduce a denaturing agent-containing buffer and sonicate to re-solubilize the protein pellet.

Finally, add a protease mix that cleaves proteins. Store the sample for further processing.

To begin, collect the neutrophil-differentiated HL60 cells in a 15-milliliter tube by centrifuging the samples at 400 times g for 5 minutes.

Wash the cell pellet twice by adding 15 milliliters of PBS and centrifuge