ReadiPrep™ Histone Extraction Kit

1. Add ReadiPrep™  Prelysis Buffer to cell pellet and incubate for 15 minutes on ice.
2. Centrifuge the cells at 13000 rpm for 15 minutes.
3. Collect the supernatant and label as “Cytoplasmic” fraction.
4. Add ReadiPrep™  Lysis Buffer to cell pellet and sonicate the sample.
5. Incubate the mix for 60 minutes on ice.
6. Centrifuge the cells at 13000 rpm for 15 minutes.
7. Collect the supernatant and add ReadiPrep™  Balance Buffer with DTT and label as “Histone” fraction.

Important: Check the buffers to see if they contain salt precipitates before use. If so, warm at room temperature or 37°C and shake the buffer until the salts are re-dissolved.

Prepare ReadiPrep™ Balance Buffer working solution by adding 10 μL of DTT solution (Component D) into 10 mL of ReadiPrep™ Balance Buffer (Component C). 

Note  We recommend making the ReadiPrep™ Balance Buffer working solution fresh as per need. Storing the ReadiPrep™ Balance Buffer working solution at <-15 °C may cause precipitation.

1. Harvest cells and pellet the cells by centrifugation at 1000 rpm for 5 minutes.
2. Resuspend the cells in 200 μL of ReadiPrep™ Prelysis Buffer and lyse the cells on ice for 15 minutes.
   Note: It is recommended to use 10^7 cells for the volume indicated above. For optimal extraction, cell number must be optimized with indicated buffer volume.
3. Centrifuge the cells at 13000 rpm for 15 minutes.
4. Collect the supernatant. The supernatant will contain the cytoplasmic fraction.
5. Resuspend the remaining cell pellet in 150 μL of ReadiPrep™ Lysis Buffer and vortex the sample.
6. Sonicate the samples at 30% power for 15 seconds.
7. Incubate the sample on ice for 60 minutes.
8. Centrifuge the cells at 13000 rpm for 15 minutes.
9. Collect the supernatant and add 90 μL of ReadiPrep™ Balance Buffer working solution.
10. Aliquot and store the histone extract at -20 °C for several days or -80 °C for long-term storage. Avoid repeated thawing and freezing cycles.