No discussions found
Start your discussion
Share your thoughts or question with experts in your field
Start a discussion
Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
|
ChIP assays were performed using the EpiQuik™ Tissue Chromatin Immunoprecipitation kit (Epigentek Group Inc.), as previously reported [13-15]. Briefly, small pieces of normal and PCOS ovarian tissues (1-2 mm3) were crosslinked with 1% formaldehyde. Cross-linking was terminated using 1.25 M glycine. The tissues and cells were added to homogenizing buffer, triturated, disaggregated, and centrifuged at 1000 g for 5 min at 4°C. After the removal of supernatants, protease inhibitors were added and the disaggregated tissue pellet was resuspended. Chromatin was sheared by sonication. Immunoprecipitation was performed at room temperature for 90 min. |
|
| Protocol tips |
| ChIP assays were performed using the EpiQuik™ Tissue Chromatin Immunoprecipitation kit (Epigentek Group Inc.), as previously reported [13-15]. Briefly, small pieces of normal and PCOS ovarian tissues (1-2 mm3) were crosslinked with 1% formaldehyde. Cross-linking was terminated using 1.25 M glycine. The tissues and cells were added to homogenizing buffer, triturated, disaggregated, and centrifuged at 1000 g for 5 min at 4°C. After the removal of supernatants, protease inhibitors were added and the disaggregated tissue pellet was resuspended. Chromatin was sheared by sonication. Immunoprecipitation was performed at room temperature for 90 min. |
| Upstream tips |
Protocol tips |
Downstream tips |
| -It is highly critical that the chromatin is of appropriate size and concentration. |
-For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.
-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
|
-Once in solution, store 1M DTT at -20°C. |
| Upstream tips |
| -It is highly critical that the chromatin is of appropriate size and concentration. |
| Protocol tips |
-For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.
-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
|
| Downstream tips |
| -Once in solution, store 1M DTT at -20°C. |
| Upstream tips |
Protocol tips |
Downstream tips |
| -Add protease inhibitors to all lysis solutions before use. |
-Use 2-25 µg for µg of chromatin.
-Keep cells on ice between the rounds of homogenisations.
- Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line. |
|
| Upstream tips |
| -Add protease inhibitors to all lysis solutions before use. |
| Protocol tips |
-Use 2-25 µg for µg of chromatin.
-Keep cells on ice between the rounds of homogenisations.
- Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line. |
Can't find the product you've used to perform this experiment? It would be great if you can help us by
Adding a product!