Isolation and Digestion of Neutrophil Proteins
Transcrição
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.
