The overall goal of this procedure is to identify protein binding regions via chromatin. This is accomplished by First cross-linking the protein to the DNA. The second step is to shear the DNA.
Next, the DNA bound target protein is immuno precipitated and then alluded. The final step is to reverse the cross-link and to isolate the DNA. Ultimately, chromatin immunoprecipitation is used to show localization of protein binding across the genome.
This method can help answer key questions In the functional genomics field, such as identifying and mapping protein DNA interactions in a given tissue or cell line, The cells for this procedure are grown in 100 by 20 millimeter cell culture dishes. The amount of cells can range from one to 10 million cells per dish, depending on cell type. Approximately 2 million cells are sufficient for one immunoprecipitation to cross-link protein and chromatin in the cells.
Add formaldehyde to 1%and incubate for 10 minutes at room temperature with occasional rocking quench cross-linking by adding glycine to a final concentration of 125 millimolar and incubating for five minutes. At room temperature, wash cells twice with one x phosphate buffered saline after the second wash, decant PBS and add 0.2 milliliters of fresh PBS harvest cells with a plastic cell scraper into a micro centrifuge tube. Spin down the cells at 2000 RPM for five minutes at four degrees Celsius.
Aspirate the supernatant for nuclear extraction. Keep the cells as a pellet for preparing a whole cell lysate, which will be demonstrated in the next segment. Resus suspend cells in SDS lysis buffer whole cell lysates are prepared from the cross-linked cells by sonication on wet ice.
Using a My ator To obtain the ideal DNA fragment size, this procedure must be optimized by trying out different sonication conditions. The samples must remain on ice throughout the procedure. Sonicate each sample at these settings, 30 seconds on and 45 seconds off at an amplitude setting of two.
In this example, 12 cycles are needed to obtain the ideal fragment size. Collect 20 microliters of each sample to check sonication results and for quantification. The rest of the sample can be stored at negative 80 degrees Celsius.
Dilute the 20 microliter sample by adding 30 microliters of 0.1 XTE buffer. Then treat the sample with one microliter of RNAs A at 37 degrees Celsius for one hour. Next, add one microliter of proteinase K and incubate at 62 degrees Celsius for two hours.
After the proteinase K treatment run 20 microliters of the sample on a 2%agros gel. The samples should present a smear with the bulk of the DNA at the desired size. If several different cycles are tested, a gradual decrease in size should be seen.
As the number of cycles increase, the remaining amount of sample will be purified with a kaya quick PCR purification kit and quantified using a NanoDrop spectra of TER to begin this procedure. Fava sonicated chromatin samples on ice centrifuge at 12, 000 RPM for 10 minutes at four degrees Celsius. Put on ice right away.
To remove the SDS, which appears as a white pellet. Collect the supernatant and discard the pellet from each sample. Combine samples if necessary, set aside the amounts needed for the experiment based on previous calculations.
One to 10 micrograms of chromatin is required per immunoprecipitation. Dilute the chromatin one to 10 inchi dilution buffer supplemented with proteinase inhibitor. Add 100 microliters of blocked aro speeds per immunoprecipitation and rotate at four degrees Celsius for one hour.
Begin the procedure for chromatin immunoprecipitation by spinning down samples at 800 RPM for one minute at four degrees Celsius, and transferring each supernatant to a fresh tube. Centrifuge the supernatant at 800 RPM for one minute and transfer to another clean tube. Save 20 microliters of the supernatant at negative 20 degrees Celsius to serve as the input control.
Later aliquot the rest of the chromatin to the number of immuno precipitations required in the experiment. To each sample, add two micrograms of antibody per one to 10 micrograms of chromatin incubate overnight at four degrees Celsius with rotation On the following morning, add 100 microliters of blocked aros beads to each immunoprecipitation sample. Incubate for one hour at four degrees Celsius with rotation after one hour.
Pellet the beads by spinning down at 800 RPM for one minute. Discard as much of the supernatant as possible. Wash beads.
Once with a low salt immune complex wash buffer, add one milliliter of the buffer to each tube. Rotate at room temperature for five to eight minutes and then centrifuge at 800 RPM for one minute after discarding the supernatant wash beads once with high salt immune complex wash buffer once with lithium chloride, immune complex wash buffer, and twice with TE buffer for a total of five washes. Subsequently, DNA is alluded from each immunoprecipitation sample as well as input samples.
To reverse the cross link, add eight microliters of five molar sodium chloride to 200 microliters of each elu and input control. Seal the tubes with param and incubate in a 65 degrees Celsius water bath overnight. On the following day, treat each sample with one microliter of RNAs, a for one hour at 37 degrees Celsius.
After an hour, add four microliters of 0.5 molar EDTA and eight microliters of one molar tris HCL to each sample and mix. Then add one microliter of proteinase K and incubate at 45 degrees Celsius for two hours. Purify the samples using the kaya quick PCR purification kit.
The samples can be saved at negative 20 degrees Celsius until they're examined by PCR. The enrichment by chromatin immunoprecipitation can be checked by either PCR or real-time PCR For PCR samples, electro referre on an agros gel, there should be bands in the input and chip sample lanes using an antibody for the protein of interest, which is the transcription factor TCF seven L two in this case. In contrast, there should be nothing or at most a very faint band in the IgG negative control lane for the positive binding region.
For the negative binding region. There should be a very faint band or no band at all for the IgG control and chip lanes. There should be a band in the input lane.
This figure shows results from the same samples examined by real-time PCR. As with the previous result. There should be a significant fold enrichment of the positive binding region for the chip sample over the IgG control.
In addition, there should be very little enrichment, if any, in the negative binding region. After watching this video, you should have a good understanding of how to cross-link binding protein to chromatin in a tissue or cell line, and meaning precipitate your targeted protein, and then isolate the DNA for further analysis.
