Hi, I'm Stefan Burani from the laboratory of Dr.Frank Zawa in the Department of Biochemistry at the University of California Riverside. Today we will show you a procedure of chromatin immunoprecipitation. So let's get started.
This procedure begins with formaldehyde cross-linking of human embryonic stem cells Prior to cross-linking. Human embryonic stem cells are thawed from liquid nitrogen for five minutes at 37 degrees Celsius, and then washed with media. The cells are then plated on gelatin coated six well plates and allowed to expand for two to three days.
For each immunoprecipitation reaction, we will need to start with 50 to 100 million cells. The cells are first ized and collected in suspension. Fresh from aldehyde is added to each cell suspension at a concentration of 1%and incubated for 10 minutes at room temperature.
Next, add one 10th volume of 1.25 molar glycine to quench the formaldehyde and incubate for 10 minutes. Wrist the cells twice with 10 milliliters of PBS and then pull em in 50 milliliter conical tubes. Spin the tubes in a centrifuge at 1000 RPM for five minutes at four degrees Celsius in order to collect the cells in a pellet.
After the spin, discard the super natin and resuspend the pellet in 1.5 milliliters of lyce buffer. Finally, transfer the cells into a 1.5 milliliter micro fuge tube. After the cells have been transferred, flash freeze them three times in liquid nitrogen and then use a tissue grinder to break them.
Once the cells are cross-linked, they're ready for immunoprecipitation or they may be stored frozen at minus 80 degrees Celsius indefinitely. Once you have cross-linked the cells, you need to prepare the magnetic beads to do this. At 50 microliters of DI beads to 1.5 milliliter lower retention micro tubes, set up one tube of beads per immune precipitation.
Then add one milliliter of block solution. Now collect the dyal beads using dyal MPC. Place the tubes in the magnetic rack.
Allow the beads to collect on the side of the tube. It should take about 15 seconds. You can invert the rack twice to help collect beads.
Once the beads have been collected, you can remove the sate with pipetter. After the supernatant has been removed, add one milliliter of block solution and gently resuspend the beads by removing the magnetic strip from the rack and inverting the rack. With the tube still in place, you can do this 10 to 20 times or until the beads are evenly.
Resuspended collect the beads as you did previously. Once collected, you can remove the supernatant with a pipetter. Repeat this wash using 1.5 milliliters of block solution.
After the second wash, you can resuspend the beads in 750 microliters of block solution and then add 10 micrograms of antibody. In our research, we use an antibody against the protein MLL incubate the mixture at four degrees Celsius for a minimum of six hours or overnight on a mutator. During this incubation, you may sonicate your crosland chromatin, which we will demonstrate shortly after the overnight incubation.
Remove the antibody solution and then wash the beads three times. Using one milliliter of lyase buffer as you did previously, using the magnetic rack to collect the beads. After the washes, you're ready to add your sated chromatin.
Before beginning sonication, you first need to retrieve the frozen cell pellets from the minus 80 degrees Celsius and allow them to thaw. Prepare a 15 milliliter conical tube for sonication. This is done by cutting the tube in two pieces at the five milliliter mark and discarding the upper half.
The tubes can be covered with param or the tube cap. While setting up. Using a tube rack position the tube so that the ator probe sits approximately 0.5 to one centimeter above the bottom of the tube.
Be sure that the probe is centered and does not contact the side of the tube to prevent foaming of the solution. Now sonicate the suspension for six rounds of 20 seconds. Each samples should be kept in an ice water bath between rounds of sonication to decrease foaming.
Initially set the power output to zero and then manually increased to the final power during the first burst. After some ation, transfer the material to a 1.5 milliliter tube and then spin at 13, 000 RPM for 10 minutes at four degrees Celsius. To pellet the debris and harvests the sna.
Transfer the SINA to a new 1.5 milliliter tube. Save 50 microliters of the cell lysate from each sample as input DNA. This will be used later to check the size of the shared chromatin.
This can be stored at minus 20 degrees Celsius. At least one input DNA aliquot should be kept per batch of sonicate lysate. Now we are ready to immuno precipitate the chromatin to amino precipitate the chromatin.
First, add the chromatin equivalent of 50 to 100 million cells from the sating collected in the previous step to 50 microliter antibody magnetic feed mix that we prepared earlier to a final volume of one milliliter. If necessary, this volume could be adjusted with lyce buffer invert to resuspend. The beads incubate this mixture overnight on the mutator at four degrees Celsius.
After incubation, collect the beads using dyl MPC place the tubes in the rack, allowing the beads to collect on the side of the tube. This should take approximately 20 seconds afterwards. Infer the rack twice to help collect all the beads.
Then remove the sedant with a pipetter changing tips between samples. Next, we need to wash the beads. To do this, add one milliliter of lyase buffer to each tube and gently resuspend the beads.
This can be done by removing the magnetic strip from the rack and inverting the rack with the tubes still in place about 10 to 20 times or until the beads are evenly resuspended. Afterwards, collect the beads by replacing the magnetic strip. Remove the ate by pipetter.
Repeat this wash three to five more times. Remember to change tips between washes. Next, wash the beads four to six times in one milliliter of IP one buffer as just described.
Then similarly, wash the beads four to six times in one milliliter of IP two buffer. Finally, wash the beads four to six times in one milliliter of TE buffer pH 8.0 as done with the other washes. Remove the TE after the final wash.
Now spin a 3000 RPM for three minutes at four degrees Celsius to remove any residual TE buffer. The beads are now ready for evolution to e remove the chromatin. First, add 210 microliters of evolution buffer and elute the material from the beads by incubating the tubes in a 65 degree Celsius thermal mixer at 800 RPM incubate for 15 minutes or up to 30 minutes to improve recovery of the eluate.
Spin down the beads at 13, 000 RPM for one minute. Our temperature after centrifugation, remove 200 microliters of the S nain and transfer to a new tube, and then add 200 microliters of TE 0.0. Now our samples are ready to CROs link to reverse crosslink deviated precipitated DNA.
Incubate the supernat in a 65 degree Celsius water bath for a minimum of six hours at a maximum of 15 hours. This incubation can be done in a water bath to minimize condensation. Next, follow the 50 microliters of input DNA.
That was reserved after sonication reverse cross length this input DNA by incubating it at 65 degrees Celsius as was done with eluate. Now we are ready to purify the DNA. The DNA is then purified and precipitated using phenyl chloroform extraction, followed by ethanol precipitation techniques.
The precipitated DNA pellets are resuspended in water and then can be stored at minus 20 degrees Celsius. For future use, the purified input DNA can be run on an agro rush gel to check the size of the shear chromatin. Ideally, the chromosome should be between 250 and 750 bases.
So we have just shown you how to do a chromatin immunoprecipitation from human embryonic stem cell. When you're doing this experiment, it's important to use a height affinity antibody chromatin with the right size range and a high stringency wash steps. So that's it.
Thank you for watching and good luck for your experiments.
