This article discusses a protocol used for chromatin preparation from tissue for the subsequent use in chromatin immunoprecipitation (ChIP).
It is recommended to use 30 mg of liver tissue for each ChIP/antibody. The amount, however, may differ in the case of other tissues. Protein abundance, the efficiency of cross-links, and antibody affinity are the factors that determine the amount of tissue to be used. For each ChIP assay, 5-15 µg chromatin was used to optimize the protocol.
It is important to determine the exact concentration of chromatin to be used for each type of tissue before initiating the X-ChIP assay. Abcam’s cross-linking chromatin immunoprecipitation (X-ChIP) protocol should be employed following the preparation of chromatin as explained in the following sections. All solutions used should include protease inhibitors, which include leupeptin 1 µl/ml, aprotinin 1 µl/ml, and PBS PMSF 10 µl/ml.
- It is recommended to thaw frozen tissues on ice. The time required for this process varies based on the amount of tissue used. The frozen tissue samples should not be heated to a high temperature to avoid degradation of samples by proteases. Samples should always be placed on ice and all steps should be quickly carried out so as to minimize thawing. Tissue cutting should be performed in a petri dish placed over a block of dry ice.
- Two razor blades are used to cut fresh or frozen tissue into tiny pieces (between 1-3 mm3)
- An empty 15 ml conical tube is weighed, the tissue is then transferred into the tube and weighed again to determine the amount of tissue
- Cross-linking solution is prepared in fume hood using 10 ml PBS per gram of tissue, followed by adding formaldehyde to a final concentration of 1.5% and rotating the tube for 15 minutes at room temperature
- Glycine is added to a final concentration of 0.125 M to stop the cross-linking reaction and the tube is rotated for another 5 minutes at room temperature
- Tissue samples are then centrifuged for 5 minutes at 720 rpm and 4°C
- Media is aspirated and washed with10 ml ice-cold PBS and then centrifuged for 5 minutes at 720 rpm and 4°C. The wash buffer is then discarded
*The tissue may be snapped frozen at this stage in liquid nitrogen and stored at -70°C. Avoid multiple freeze-thaws. If using immediately, resuspend tissue in 10 ml cold PBS per gram of starting material. Place on ice.
- Becton Dickinson’s Medimachine may be employed to produce a single cell suspension. Two medicones (50 µm) per gram of tissue is used to process.
- A 1 ml pipette tip is cut to make the orifice larger
- Tissue resuspended into 1 ml of PBS is added between 50-100 mg (3-4 chunks)
- This solution is added to the medicone and the tissue is ground for 2 minutes
- The cells from the medicone are collected by inserting a 1 ml syringe and an 18 gauge blunt needle. Then the cells in conical tube placed on ice are collected.
- Step 2 is repeated until the all of the tissues are processed
- A microscope is used to check the cell suspension to ensure the formation of a unicellular suspension. If more grinding is required, more PBS is added to the tissue and Steps 2 to 5 are repeated until grinding all the tissue into a homogeneous suspension
- The cells are then centrifuged for 10 minutes at 1000 rpm and 4 °C. The cell pellet volume is measured for next step
- Supernatant and resuspend pellet in FA Lysis Buffer (750 μl per 1x107 cells) are carefully aspirated off
- The X-ChIP protocol is continued from the sonication step
- 50 mM HEPES-KOH pH7.5
- FA Lysis buffer
- 1 mM EDTA pH8
- 140 mM NaCl
- 0.1% Sodium Deoxycholate
- 0.1% SDS
- 1% Triton X-100
- Protease Inhibitors (add fresh each time)
This article was adapted from protocols provided by Peggy J Farnham, Luis G. Acevedo, and Henriette O'Green.
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