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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
-Warm IP Lysis Buffer to room temperature to prevent precipitation.
-Prepare one extra plate of cells to estimate cell number.
-Thaw Protease Inhibitor Cocktail (PIC) at room temperature.
-It is recommended to take some trial to optimize sonication condition before beginning with sonication. |
- In general it is recommended that one million mammalian cells are required for each IP
fraction.
-Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles.
-To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight. |
|
| Upstream tips |
-Warm IP Lysis Buffer to room temperature to prevent precipitation.
-Prepare one extra plate of cells to estimate cell number.
-Thaw Protease Inhibitor Cocktail (PIC) at room temperature.
-It is recommended to take some trial to optimize sonication condition before beginning with sonication. |
| Protocol tips |
- In general it is recommended that one million mammalian cells are required for each IP
fraction.
-Centrifuge the sample at 6000 × g for 5 min at 4 °C to eliminate bubbles.
-To reduce background, remove as much supernatant as possible in the last wash. Continue with next step OR store at 4°C overnight. |
| Upstream tips |
Protocol tips |
Downstream tips |
-Optimal conditions to obtain the desired fragment size should be
determined prior to the immunoprecipitation (IP) by
performing a sonication time course. |
-The minimum number of cells to be processed is 3x106 cells for 3 ChIPs.
-Sonicating for too long will disrupt nucleosome-DNA
interactions therefore the band size should not be smaller than 200 bp.
-The amount of antibody can vary but 2-5 µg is a good starting point. |
|
| Upstream tips |
-Optimal conditions to obtain the desired fragment size should be
determined prior to the immunoprecipitation (IP) by
performing a sonication time course. |
| Protocol tips |
-The minimum number of cells to be processed is 3x106 cells for 3 ChIPs.
-Sonicating for too long will disrupt nucleosome-DNA
interactions therefore the band size should not be smaller than 200 bp.
-The amount of antibody can vary but 2-5 µg is a good starting point. |
| 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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