The Isolation and Digestion of Proteins from Neutrophils
Transkript
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 again at 400 times g for 5 minutes.
Add 300 microliters of ice-cold 100-millimolar Tris HCl pH 8.5 containing a proteinase inhibitor cocktail tablet to each sample, and vortex them briefly.
Then, add 1 by 10 volume of 20 percent SDS to a final concentration of 2 percent and probe-sonicate the samples in an ice bath for 30 seconds on and 30 seconds off, for five cycles at 10 percent power.
To collect the spray-off from the tube walls, centrifuge the samples at maximum speed for 5 to 10 seconds, not letting the cell debris form a pellet.
Transfer the samples from the 15-milliliter tube into a 2-milliliter tube and add 1 by 100 volume of 1 Molar stock Dithiothreitol to a final concentration of 10 millimolar.
After vortexing briefly, incubate the samples at 95 degrees Celsius for 10 minutes with shaking at 800 rpm. Then, cool the samples to room temperature by placing them on ice.
Next, in dark conditions, add 1 by 10 volume of 0.55 Molar Iodoacetamide to a final concentration of 55 millimolar, and vortex the tube before incubating at room temperature in the dark for 20 minutes.
At the end of incubation, add 100 percent ice-cold acetone to a final concentration of 80 percent. Store the samples at minus 20 degrees Celsius overnight. Store them for up to two weeks for later use.
The following day, centrifuge the samples at 13,500 rpm for 10 minutes at 4 degrees Celsius to precipitate the cell pellet. Wash the cell pellet twice by adding 500 microliters of 80 percent ice-cold acetone, followed by centrifugation for 10 minutes at 13,500 rpm and 4 degrees Celsius.
To the air-dried cell pellet, add 100 microliters of 8 Molar Urea in 40 millimolar HEPES, and water-sonicate in a 4-degree Celsius water bath for 30 seconds on and 30 seconds off for 15 cycles to resolubilize the protein pellet before quantifying the protein.
Next, add 300 microliters of ammonium bicarbonate to dilute the sample to a final concentration of 2 Molar Urea. To digest 50 micrograms of protein, add Trypsin and lysine-C to the samples on the ice at an enzyme-to-protein ratio of 1 to 50.
Mix the samples by gentle tapping and incubate them overnight at room temperature for digestion. Then, proceed to the peptide purification protocol.
