Trouble with subcellular fractionation - (Apr/26/2008 )
Hello,
actually I have found a good protocol for fractionation of cytoplasm and nuclei. In particular I turn my attention to the nuclei. But lately I always got cytoplasmic contamination in the nuclei fraction.
Method briefly described:
lysis with a buffer (0.1%TX100),
dounce 10 strokes,
500g 6 min,
supernatant discard,
pellet resuspends in buffer and layers over sucrose buffer (45%),
1600g 30 min, pellet wash with surcose buffer (10% + MgCl2),
dounce 7 strokes, 500 g, 6 min,
Pellet takes up in buffer.
I have prepared all buffers new. No success.
I take equal amounts of sucrose buffer and resuspended nuclei solution at the sucrose gradient. Shall I repeat the gradient or shall take more sucrose buffer? Please help me, I am really despaired. Where could be the leakage of my doing?
Thanks a lot
Tiffy
actually I have found a good protocol for fractionation of cytoplasm and nuclei. In particular I turn my attention to the nuclei. But lately I always got cytoplasmic contamination in the nuclei fraction.
Method briefly described:
lysis with a buffer (0.1%TX100),
dounce 10 strokes,
500g 6 min,
supernatant discard,
pellet resuspends in buffer and layers over sucrose buffer (45%),
1600g 30 min, pellet wash with surcose buffer (10% + MgCl2),
dounce 7 strokes, 500 g, 6 min,
Pellet takes up in buffer.
I have prepared all buffers new. No success.
I take equal amounts of sucrose buffer and resuspended nuclei solution at the sucrose gradient. Shall I repeat the gradient or shall take more sucrose buffer? Please help me, I am really despaired. Where could be the leakage of my doing?
Thanks a lot
Tiffy
do you use a loose-fit potter for initial homogeneization?
leave triton x-100, it also disintegrates the nuclear envelope; you need no lysis if you homogeneize; carefully check osmolarity of your initial homogeneization buffer, start with 300 mosM
I use a homogenizer Dounce Pestle A and the components of the lysis buffer in which I dounce are 10 mM Tris, pH 7.5; 10 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Triton X-100 and protease inhibitors. Could I omit the homogenization rather then the Tx100, because the procedure is not constant. I mean I don`t do it in the same manner. Sometimes I exert more strength on the homogenization than at other runs. You mention 300mM. To what does it refer to? Presumably NaCl.
Tiffy
Tiffy
your lysis buffer should be substituted by a isoosmolaric homogeneization buffer; you use a hypoosmolaric lysis buffer which is not ideal to isolate intact nuclei
Hello The Bearer,
I must excuse for my ignorcence but what is actually the advantage of an isoosmolaric buffer compare to hypoosmolar? Okay I know that the cytoplasmic microenvironment is isoosmolaric, isn`t it? Could you briefly describe it. That would be very kind. Or where can I look it up.
Tiffy