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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
-If you are unfamiliar with cell type being used, culture an extra dish of cells for determining cell number.
Before crosslinking, trypsinize and determine the cell number from the extra dish of cells. |
-After incubation, it is essential to open the column cap before plug removal to equalize the pressure within the column. Failure to open the cap before plug removal will result in sample loss. |
-If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use. |
| Upstream tips |
-If you are unfamiliar with cell type being used, culture an extra dish of cells for determining cell number.
Before crosslinking, trypsinize and determine the cell number from the extra dish of cells. |
| Protocol tips |
| -After incubation, it is essential to open the column cap before plug removal to equalize the pressure within the column. Failure to open the cap before plug removal will result in sample loss. |
| Downstream tips |
| -If you are preparing chromatin in bulk, unused supernatant may be stored at -80°C for later use. |
| Upstream tips |
Protocol tips |
Downstream tips |
| -Do not use formaldehyde if white precipitate is visible in the solution. |
-Use ChIP validated antibody.
- 1X104 cells per ChIP when using high affinity antibodies directed towards more abundant proteins.
-Protease Inhibitor Cocktail III contains DMSO and will remain frozen below 18.4°C.
-Keep cell lysate ice cold. Sonication produces heat, which can denature the chromatin. Allow at least 30
seconds between cycles of sonication to prevent sample overheating.
|
|
| Upstream tips |
| -Do not use formaldehyde if white precipitate is visible in the solution. |
| Protocol tips |
-Use ChIP validated antibody.
- 1X104 cells per ChIP when using high affinity antibodies directed towards more abundant proteins.
-Protease Inhibitor Cocktail III contains DMSO and will remain frozen below 18.4°C.
-Keep cell lysate ice cold. Sonication produces heat, which can denature the chromatin. Allow at least 30
seconds between cycles of sonication to prevent sample overheating.
|
| Upstream tips |
Protocol tips |
Downstream tips |
|
-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation.
Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody.
-Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target. |
|
| Protocol tips |
-Just because an antibody works well in a Western blot does not always indicate it will perform well in chromatin immunoprecipitation.
Unlike a Western blot that detects proteins that have been denatured, a ChIP antibody must recognize the target protein in its native state. Use ChIP-validated antibody.
-Use 2-10 μg of your ChIP antibody depending on the abundance of your protein target. |
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