The overall goal of this procedure is to accurately determine the relative levels of post-translational modifications within preparations of total cellular nucleosomes obtained from a million cells. This is accomplished by first preparing high quality homogenous cultures of mammalian cells. Then nuclei are isolated from the cells through mechanical disruption with a downs, homogenizer and centrifugation.
The next step is to obtain intact nucleosomes by digesting the chromatin present within the nuclei and performing a series of hypotonic extractions. Lastly, the nucleosomes are interrogated using an ELIZA assay with the appropriate controls to identify specific post-translational modifications. Ultimately, results can be obtained that show the relative levels of specific modifications present within nucleosome samples.
The main advantage of this technique over existing methods like western blotting and chromatin immunoprecipitation is that a global picture of a number of nucleosome modification states can be readily determined. The method is much more sensitive and accurate than western blotting and can be performed in most labs equipped for general molecular biology experimentation. This method can help answer key questions in the stem cell and epigenetics field, such as what happens when major differentiation and reprogramming events occur.
To begin the procedure, first tryps anize the cells with three milliliters of trypsin per 15 centimeter plate to freshly tryps inized cells. Add 20 milliliters of ice cold PBS butyrate. Transfer the cells to a 50 milliliter conical tube and centrifuge the tube to collect the cells at 1000 RPM for five minutes, aspirate the supernatant and add 10 milliliters of ice cold PBS butyrate to wash centrifuge the cell suspension again at 1000 RPM for five minutes.
Remove the supernatant and resuspend the cell pellet in four milliliters of lysis buffer with protease inhibitors as described in the written protocol. Next, pipette the cells to A type B pestle down homogenizer on ice down homogenize with 20 strokes. Then transfer the homogenate to a centrifuge tube on ice.
Centrifuge the sample at 2000 G for 10 minutes. At four degrees Celsius. Remove the super named, which contains the cytoplasmic material and resus.
Suspend the pellet in two milliliters of ice cold wash buffer C With protease inhibitors, gently layer the resuspended material onto a five milliliter, 30%sucrose cushion in a cold centrifuge tube. To isolate the nuclei centrifuge at 2, 400 G for five minutes in a swinging bucket, rotor nuclei will migrate through the cushion and cellular debris remains at the interface. After centrifugation, carefully remove all liquid volume and re suspend the nuclei in 250 microliters of ice cold wash buffer C with protease inhibitors, transfer the nuclei to a 1.5 milliliter micro centrifuge tube.
To begin isolation of nucleosomes, add three microliters of 0.1 molar calcium chloride to the nuclei and place in a 37 degree Celsius heat block until the temperature is equilibrated. Then add two units of micro cocal nuclease in 10 microliters of micro cocal nuclease, buffer, and incubate for 12 minutes at 37 degrees Celsius. Mix frequently with a pipette tip.
After the incubation, stop the reaction with six microliters of 0.5 molar sodium E-D-T-A-P-H eight, and place on ice to cool. Next centrifuge the sample at 2000 G for four minutes following centrifugation, discard the snat and resuspend the pellet. In 300 microliters of 0.2 millimole or sodium EDTA, incubate the sample on ice for one hour with occasional gentle pipetting to mix.
Following a centrifugation at 3000 G for four minutes. At four degrees Celsius, collect the supernatant, which contains the free nucleosomes in a new Fuge tube and store on ice deliberate additional nucleosomes reus. Suspend the pellet in 300 microliters of 0.2 millimole or sodium EDT as before and incubate on ice for one hour with occasional gentle mixing.
After the incubation, centrifuge the sample. Again, combine the resulting snat with the remaining sample in the micro fuge tube on ice. To begin the nucleosome Eliza assay load a 96 well Eliza plate with a descending series of five two-fold serial dilution of nucleosomes and coating buffer, starting with 0.1 micrograms per 50 microliters in the top well all dilution series should be loaded in triplicate.
Remember to include wells with coating buffer only as controls. Incubate the plate overnight at four degrees Celsius the next day. Use a plate washer to wash the plates with 200 microliters per well of 0.5%between 20 in PBS at room temperature four times for a combined total of 10 minutes.
Now add 100 microliters per well of blocking solution and incubate for one hour at room temperature on a rotator. After the incubation, remove the blocking buffer by shaking the inverted plate briskly over a sink. Next, add the diluted primary antibody in a volume of 50 microliters per well in a solution of 5%PSA in 0.05%between 20 in PBS incubate the solution for one hour on a rotator at room temperature.
After the primary antibody incubation, wash the plates as before using the plate washer. Once the wash procedure as completed, add 50 microliters per well of horseradish peroxidase conjugated secondary antibody diluted in PBS with 5%BS, A, and 0.5%20 incubate room temperature for one hour on a rotator following secondary antibody incubation. Wash the plates as before.
Using the plate washer to develop the plate. Add 50 microliters of TMB Eliza substrate to each well and incubate for 10 minutes. At room temperature, the wells of the ELIZA plate will turn blue and the intensity is proportional to the amount of nucleosome modifications present within each.
Well then stop the reaction. By adding 50 microliters per well of two normal sulfuric acid. The color will change to brown centrifuge the plate at 1500 RPM for two minutes to dissipate any air bubbles.
The plate is now ready for quantification on a color metric plate reader. Finally, read the plate at 450 nanometers and export the results for analysis. Once mastered, this technique can be done in two days if it's performed properly After its development.
This technique opened the door for researchers in the field of stem cell research to explore epigenetic responses to differentiation and reprogramming events at the level of the entire epi proteome.
