Cell fractionation problem - Nucleaus forms a pellet that can't be suspended (Jun/27/2008 )
Hello everyone, I am trying to fractionate cell cytoplasm and Nucleus for protein quantification. I am working with HL-60 cells and the Cell lysis buffer I am using consists of 10mM Nacl, 1.5mM MgCl2, 0.05%NP40, Hepes buffer, water and protein inhibitors. After centrifugation at 1500Xg, I remove the cytoplasmic fraction but the rest of the cell forms a pellet that is hard to deal with. I can't get it to form a suspension and therefore I can not separate the proteins in thee nucleas from the dna and so on. There is little or no change when I add a hypotonic buffer. Does anyone know why this happens, the reason behind it and a possible solution? Thanks in advance.
That pellet is no longer cells, they are nuclei (and in your case, damaged ones).
Any time a nuclear membrane is damaged, the sticky chromatin, well, sticks with each other and forms a pellet, and can not be re-suspended.
You need to troubleshoot from this angle. (detergent, buffer, vortex, syringing, temp, pipetting etc)
I usually just vortex my pellet or resuspend with a wide pipette tip (or you can cut the tip off of a regular one). I've found that fractionating these cells isn't easy. Also try swelling the cells in hypotonic buffer for 15-20 minutes before adding the NP-40. I was having lots of trouble getting clean fractionation with my suspension cells, and this has helped a lot.
You will need to add more detergent to solubilize the nucleus. For example, 1% NP40 should solubilize the nuclear membrane, then you can pellet the DNA on a benchtop centrifuge at full speed (4 degrees). Make sure you have enough volume, otherwise the proteins will stick to the DNA. If you have a sonicator, you can also sonicate to shear the DNA and then spin.
By the way, you didn't mention breaking open the cells? We normally don't use any detergent (just hypotonic buffer). After swelling, break open cells with dounce homogeniser or passing through needle.
Hello everyone, thanks for all the answers. From what you said, I realized that I was treating the cells too harshly and prematurely breaking the nucleus membrane. I am now softer on them by resuspending with a wide pipette tip instead of vortexing and I swell the cells in hypotonic buffer for 10 minutes before adding the NP-40. And centrifuging immediately . I added 3.3% NP40 which seems to break just the cell membrane and not the nucleus one provided I centrifuge immediately, remove the supernatant and add more hypotonic solution to wash. I am usinging the NP40 surfactant to break open cells and I am passing through a needle after leaving on ice in a hypertonic medium for 20 minutes to break the nucleus membrane. It seems to be working well. I appreciate all who helped .
well assudi glad that it worked well for you. leukemic cells are hard to handle i understand. if u can manage to lyse the cells hypotonically and then get the sticky nuclear pellet, one wash the nuclear pellet twice in the hypotonic lysis buffer, then add appropriate volume of the lysis buffer with detergent and sonicate!!! (unless you want to do an IP with the nuclear extract)
sonication increases your net protein yield dramatically