QUOTE (Georgechao)
1. what does the first paragraph mean (Sometimes, i get part of pellet in the eppy...)? Is it important? Eppy stands for eppendorf tube, right?
i do the extraction in 1.5ml tube. after having all done, i meant i'm concerned by the fact i may have over pipett, as in cytoplasmic fraction as well for nuclear proteins fraction. So i spin my thawed tube quick before pipetting 1µl for bradford assay.
QUOTE (Georgechao)
2. in the next few steps, you pellet the cell twice (first by 1000rpm, thentransfer the cell pellet into eppys and pellet again by 2000rpm). Is it important to do everything in eppys? As I have large vol, of cell, can I use 15ml conical tube? What is the rcf of 1000rpm for the rotor that you used?
well i meant these are speeds used with my centrifuge. I do this in 1.5 ml tube
as well as 15 or 50ml tubes (falcon ubes for ex)
QUOTE (Georgechao)
3. Buffer A: PIC stands for protease inhibitor cocktail, right?
yep
. I'm not using it any more and that's no problem
cost saving for me, but you can put it if you can afford it
QUOTE (Georgechao)
4. What is the stock concentration of NP40 buffer that you used? Should I also add it drop by drop to the buffer A after you resuspend the cell pellet? I think drop by drop while vortexing could be very helpful to break down cells.
i use a 10% NP40 stock solution. I was doing 0.05% (so 1/200 dilution, but for flag affinty purification i go for 0.1%
-fred_33-
Thanks for the note.
Followup question:
After you add the NP40 (0.1%) in the buffer A (hypotonic buffer), do you let the cell resuspension sit on ice for 10min? or immediately spin down after adding NP40? I now think the vortex and NP40 in buffer A is important as I work on large amount of cells and the myotubes have tons of cytoskeleton proteins, which may interfere the nuclei exposure and lysed by high salt.
If I use NP40 for lysing the cell in buffer A, do I still need Dounce Homogenizer? I am thinking to use both. Is it reasonable?
Another thing that bothers me: after spin down the final nuclear lysate (20000g), there is always a layer of lipid stuff on the top of the supernatant. How to get rid of it? I tried to filter with 0.45u syringe filter, but the filter was stuck after half ml of solution passed. any suggestion? If ultracentrifuge is used, how high the speed should be?
Here is what I am thinking to do:
1. after getting the cell pellet, resuspend it in buffer A (4x vol of cell pellet). Resuspend it really well by pipetting. Let the cells sit for 15 min on ice.
2. While vortexing, add NP40 to final of 0.1%. ----The vortex for another 10 sec.
3. Spin down 2500g for 15 min.
4. For the pellet, add 4X of buffer A again, resuspend.
5. Dounce homogenize for 10 times.
6. Pellet again with 2500rpm for 15 min.
7. Then add buffer B. Homogenize it.
8. Then add buffer C........
Make any sense?
QUOTE (fred_33 @ Jun 7 2007, 01:42 AM)
QUOTE (Georgechao)
1. what does the first paragraph mean (Sometimes, i get part of pellet in the eppy...)? Is it important? Eppy stands for eppendorf tube, right?
i do the extraction in 1.5ml tube. after having all done, i meant i'm concerned by the fact i may have over pipett, as in cytoplasmic fraction as well for nuclear proteins fraction. So i spin my thawed tube quick before pipetting 1µl for bradford assay.
QUOTE (Georgechao)
2. in the next few steps, you pellet the cell twice (first by 1000rpm, thentransfer the cell pellet into eppys and pellet again by 2000rpm). Is it important to do everything in eppys? As I have large vol, of cell, can I use 15ml conical tube? What is the rcf of 1000rpm for the rotor that you used?
well i meant these are speeds used with my centrifuge. I do this in 1.5 ml tube
as well as 15 or 50ml tubes (falcon ubes for ex)
QUOTE (Georgechao)
3. Buffer A: PIC stands for protease inhibitor cocktail, right?
yep
. I'm not using it any more and that's no problem
cost saving for me, but you can put it if you can afford it
QUOTE (Georgechao)
4. What is the stock concentration of NP40 buffer that you used? Should I also add it drop by drop to the buffer A after you resuspend the cell pellet? I think drop by drop while vortexing could be very helpful to break down cells.
i use a 10% NP40 stock solution. I was doing 0.05% (so 1/200 dilution, but for flag affinty purification i go for 0.1%
-Georgechao-
