EpiQuik™ Total Histone Extraction Kit for Tissues ( Treated and Untreated
Re - suspend cell / tissue pellet in 3 volumes ( approximately 200 µl / 107 cells or 100 mg of tissue ) of Lysis Buffer and incubate on ice for 30 min .
Centrifuge at 12,000 rpm for 5 min at 4°C and transfer the supernatant fraction ( containing acid - soluble proteins ) into a new vial .
Prepare Balance - DTT Buffer by adding DTT Solution to Balance Buffer at a 1 : 500 ratio ( e . g . , 1 µl of DTT Solution + 500 µl of Balance Buffer ) .
Add 0.3 volumes of the Balance - DTT Buffer to the supernatant immediately ( e . g . , 0.3 ml of Balance - DTT Buffer to 1 ml of supernatant ) .
Quantify the protein concentration with an OD reading .
BSA can be used as a standard .
Aliquot and store the extract at –20°C for several days , or –80°C for long - term storage .
Avoid repeated thawing and freezing .
Weigh the sample and cut the sample into small pieces ( 1 - 2 mm3 ) with a scalpel or scissors .
Transfer tissue pieces to a Dounce homogenizer .
Dilute 10X Pre - Lysis Buffer into 1X Pre - Lysis Buffer with distilled water at a 1 : 10 ratio ( e . g . , 1 ml of 10X Pre - Lysis Buffer + 9 ml of water ) .
Add the Diluted 1X Pre - Lysis Buffer at 1 ml per 200 mg of tissue , and disaggregate tissue pieces by 50 - 60 strokes .
Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 min at 4°C .
Remove supernatant .
If total mixture volume is less than 2 ml , transfer mixture to a 2 ml vial and centrifuge at 10,000 rpm for 1 min at 4°C .
