how to do it right???
After acid extraction, the histone has to be quantified before running Western
I followed a paper in Nature Methods and run gel using 1, 3, and 5ul of samples and try to compare between different samples, like:
1ul of Sample A is about = 3ul of Sample B...
This is nowhere close to quantitative, and ususally need to do it more than 1 time, especially when you have a lot of samples...
then I saw some paper using micro BCA
so I tried it, like normal BCA, give you very precise values....
but then when I follow that to load "equal" amount of histone and do western, I later realize the histones I studied are in fact not very normalized in different lanes , probably because the micro BCA measure all protein in the samples, and you can have inpurities and varying ratios of different histones
like, even if the BCA is correct, it is possible that:
sample A = 1ug H1 + 2ug H2A + 2ug H2B + 3ug H3 + 1ug H4 + 1ug others = 10ug
sample B = 1.5ug H1 + 2ug H2A + 2.5ug H2B + 2ug H3 + 1ug H4 + 1ug others = 10ug
then if I look at H1 or H2B or H3, it will not be normalized
so, having said that, I have NO IDEA how to quantify the samples well in a convenient way
Any thoughts????
0
No replies to this topic
