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I want to detect potential changes in histone acetylation in different tissues in mice after treatment with different types of substances with HDAC inhibitory actions and I'm considering using your Episeeker kits for isolation of histones and quantification of changes in histone acetylation.
I have a few questions about these procedures:
1. Is it possible to freeze pieces of tissue/cell pellets before running them in the histone extraction kit?
2. Since I'm using mice and want to look at histones not only from tissue but also from leukocytes I will have limited number of starting cells (probably < 10^6). Is this a problem? Would it be better to pool blood to get more cells?
3. Which method/protocol do you recommend for quantifying protein/histones after the histone extraction procedure to make sure equal loading of sample in the histone H3 or H4 total acetylation detection kit?
Asked on Jan 14 2014
1. Yes, it is possible for tissue or cell pellets to be frozen before extracting histone proteins.
2. The minimum amount of histone protein required as input for these assays are 50 ng (both for the colorimetric and fluorometric kits). In principle, 50 ng of histone proteins could be obtained from as little as 100,000 cells so pooling of blood may not be necessary. However we recommend using 1 million cells if possible in order to obtain a suitable amount of histone proteins for the assay.
3. We recommend using any standard protein quantification assay such as a Bradford assay for histone quantification.
Elisa ThomasAbcam Scientific Support
Answered on Jan 14 2014