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https://www.abcam.com/Histone-Acetyltransferase-Activity-Assay-Kit-Colorimetric-ab65352.html My sample is histone which is extracted with acidic buffer. This acidic buffer includes DDT so, would like to ask how can it remove be removed from the histone sample. If you have a protocol for this situation, or whole protocol for sample preparation, please let me know.
Asked on Aug 12 2011
DDT should be avoided, as this compound strongly interferes with the reactions in the kit. There are a few things I would like to suggest: - After acid extracting the histones in your current protocol, the sample could be dialysed overnight in 1 x TBS with 3,500 MWCO dialysis tubing. This would neutralize the sample. - You could use our nuclear fractionation protocol, but exclude DTT from the buffers. Here is a link to the protocol: https://www.abcam.com/index.html?pageconfig=resource&rid=11408 - Our cell fractionation kit, ab109719 Cell Fractionation Kit Standard, could be used: https://www.abcam.com/index.html?datasheet=109719 The nuclear extraction buffer in this kit contains SDS and DTT to generate a nuclear fraction for WB. Therefore this fraction is not compatible with a subsequent enzymatic analysis (though the other fractions are active). However, it may be possible to analyze the nuclear fraction without adding this buffer. Simply add water to the N fraction or treat with RIPA buffer : 1X RIPA Buffer: 20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate 2.5 mM sodium pyrophosphate 1 mM b-glycerophosphate 1 mM Na3VO4 1 μg/ml leupeptin
Answered on Aug 12 2011