Hi,

Procedure to isolate total histones:

1) cell pellets were resuspended in 1 ml of hypotonic solution.

2) The nuclei were pelleted and sulfuric acid (0.2 M) was added to the pellet to extract the histones.

3) 20% TCA was added to the supernatant in order to precipitate histones.

4) Wash pellet with cold acetone and dry pellet by placing tube at 95°C.

5) Dissolve the pellet in hypotonic solution, measure the protein concentration.

From HaCaT cells, isolated total histones using above method followed by performed western blot to determine the histone 1.2 expression level changes. Ponceau stained PVDF membrane (attached image) shows Lamin B around 66 kDA, if I am correct and western blot shows desired band at 32 kDa. May i use lamin B as a loading control for my studies? if not which loading control shall i use for my studies?

TIA