No replies to this topic

[pakbiochemist]

Dear friends,

I have extracted histones from human tissues and have followed the following steps (Hake B, 2007)

1. PBS washing
2. Homogenization in lysis buffer (Tris, KCl, MgCl2, DTT, PMSF, Protease inhibitor, phosphatase inhibitor)
3. Centrifuge at 10,000g (10')
4. Pellet suspended in 0.4N H2SO4
5. Overnight incubation
6. Centrifugation at 16,000g (10')
7. Supernatant precipitated with TCA
8. Centrifuge at 16,000g (10')
9. pellet washed with acetone

My supervisor is saying that i cannot extract histones like this without first separating nuclei  asmost of the histone extraction protocols uses sucrose gradientsfor nuclei isolation. Please guide me what i have extracted through this. Is this a crude extract or what?


Binsan
PhD Student