This protocol is significant, because it provides a method to separate four cellular compartments from one another, the cytoplasm, the nucleus, the mitochondria, and the membrane. Not only that, but it's a scalable, reproducible procedure that has reliable results. The major advantage of this technique is the separation of four cellular compartments with minimal usage of detergents, which allows for a much more reproducible, reliable fractionation procedure.
For cytosolic protein isolation, grow U937 cells in RPMI 1640 with 10%fetal bovine serum at 37 degrees Celsius and 5%carbon dioxide to a final total of six by 10 to the eighth cells. Centrifuge the culture, and resuspend the cell pellet in room temperature PBS to a final concentration of four by 10 to the sixth cells per milliliter, pipetting gently to break up clumps. After a second centrifugation, resuspend the pellet in ice cold lysis buffer A at a final concentration of two by 10 to the seventh cells per milliliter.
Add 7.5 milliliters of cells to a conical tube on ice for downstream nuclear protein extraction, and pellet the remaining cells by centrifugation. Resuspend the pellet in cytoplasmic isolation buffer at a final concentration of two by 10 to the seventh cells per milliliter, pipetting gently to break up clumps, before incubating the cells for 20 minutes at four degrees Celsius with end over end rotation. At the end of the incubation, centrifuge the cell suspension and transfer the supernatant to a clean centrifuge tube.
Centrifuge the supernatant to sediment the cellular debris. After centrifugation, transfer the supernatant to a new centrifuge tube, and repeat the centrifugation sequence until no pellet can be observed. When the sample is clear of debris, store the cytosolic fraction-containing supernatant for up to one month at four degrees Celsius.
Then resuspend the stored cell pellet in lysis buffer A to a final concentration of four by 10 to the sixth cells per milliliter. For cell homogenization, centrifuge the cell suspension and discard the supernatant to remove excess digitonin and systolic contaminants. Resuspend the pellet in ice cold cell homogenization buffer at a final concentration of four by 10 to the sixth cells per milliliter for a 30-minute incubation on ice.
Meanwhile, for bead-based mechanical lysis, add 30 grams of pre-washed stainless steel 3.2 millimeter beads to 15 milliliters of lysis buffer B in a 50 milliliter skirted tube on ice to chill. At the end of the incubation, replace the buffer in the skirted tube with cell suspension and blend the cells for five minutes at speed eight. After lysis, transfer the homogenate to a clean tube.
Centrifuge the homogenate, then transfer the supernatant to a new tube. To remove any remaining debris from the sample, centrifuge the homogenate three times as indicated, transferring the supernatant to a new tube after each centrifugation. After the last centrifugation, to isolate the crude mitochondrial fraction, transfer the supernatant to a new tube for centrifugation.
After centrifugation, transfer the supernatant to a new tube and place the crude mitochondrial fraction-containing pellet on ice. To isolate the membrane fraction, centrifuge the collected supernatant, and after discarding the supernatant place the crude membrane fraction-containing pellet on ice. For isopycnic density gradient purification, resuspend the crude mitochondrial and membrane fraction pellets in 200 microliters of lysis buffer B, and 1.8 milliliters of iodixanol.
To create a discontinuous iodixanol gradient for each sample, first, add one milliliter of 15%iodixanol to the bottom of one eight milliliter open-top, thin-walled ultracentrifuge tube per sample. Next, sequentially underlay one milliliter of 20%iodixanol, one milliliter of 25%iodixanol, one milliliter of 30%iodixanol, and one milliliter of 35%iodixanol below the 15%iodixanol layer in each tube. Add two milliliters of the crude mitochondrial pellet suspension under the bottom layer of one gradient, and two milliliters of the crude membrane pellet suspension under the bottom layer of the second gradient.
Carefully layer one milliliter of 10%iodixanol onto the top of each gradient and balance the tubes within 10 micrograms of each other by the addition of 10%iodixanol as necessary before purifying the mitochondrial and membrane fractions by density gradient centrifugation. At the end of the separation, use a needle to puncture the side of the thin-walled tubes to collect the visible bands from each sample. For nuclear protein isolation, centrifuge the 7.5 milliliter aliquot of cells suspended in lysis buffer A, and resuspend the pellet in 800 microliters of ice cold cell solubilization buffer with pipetting and vortexing.
Incubate the suspension on ice for 30 minutes to disrupt the plasma and organelle membranes, while protecting the nuclear proteins. At the end of incubation, centrifuge the cell suspension and resuspend the pellet by gently pipetting in 800 microliters of ice cold nuclear lysis buffer supplemented with one unit per microliter of Benzonase. Incubate the mixture on an end over end rotator at four degrees Celsius to disrupt the nuclear membrane.
After 30 minutes, sonicate the mixture three times for five seconds at 20%power with a five-second pause between pulses to shear the nucleic acids. After the last sonication, centrifuge the homogenate and collect the supernatant as the nuclear fraction without disturbing the pellet. Western blot of each of the fractions after density gradient purification shows localization of the mitochondrial fraction to the 25 and 30%iodixanol layers, and localization of the membrane fraction to the 10 and 15%iodixanol layers.
Western blood analysis also confirms that each isolated sample is pure and free from contamination by proteins from other parts of the cell. In addition, densitometric analysis of the band intensity of each sample confirms the reproducibility and statistical significance of the data. Improper execution of the fractionation can result in cross-contamination of the cellular components.
A high concentration of Histone H3 in the cytosolic fraction, for example, can be due to improper clarification of the cytosolic fraction. Failure during the isopycnic density purification can result in contamination of the membrane fraction. It can be useful to perform a protein quantification assay before the Western blood analysis to confirm that the fractions do indeed contain protein to allow gel loading based on the protein quantity, and to confirm that the procedure has been properly executed.
It is critical that the cytoplasmic fraction not contain a pellet. Western blots, or ELISAs may be performed following this procedure to allow you to analyze the subcellular location of different proteins under certain conditions. This technique allowed us to analyze the trafficking of different cell death factors under conditions of hyperglycemia.
And so, for us, this helped to shed light on the mechanism of the hyperglycemic shift from apoptosis to necrosis.
