Experimental set up:
HeLa cells treated with 1 µM Dexamethasone (Dex) for 6h.
IP was performed with 4.0 µg rabbit anti-GR antibody (H-300, Santa Cruz).
Recommendations for product and protocol optimization:
- Step A2
Spin cells for 5 min instead of 10 min at 500 g at 4°C (not RT) for saving time and to avoid aberrant DNA interactions that might occur if time between harvest and cross linking is too long.
- Step B8
Using a sonication device with a sounding rod with a total lysate volume of 100 uL (3 ChIP assays) is difficult to handle. As a consequence, you easily lose much sample by dispersion. Better incubate cell pellet in the indicated 100 uL buffer D/PI mix for 5 min on ice and add 1X ChIP buffer (containing PI) to a volume of 350 uL. After sonication, add 580 uL 1X ChIP buffer (incl. PI) to the lysate (step D2). From this step the total volume for each sample will be as indicated in the manual. Sonication of bigger sample volume will not compromise the outcome of your experiment!
- Step B11
Spin sonicated lysates for 10 min instead of 5 min at 14,000 g at 4°C.
General remark:
Switching temperatures from spinning to RT at step A2 and A3 does not change the experimental results. Better keep 4°C constantly.
HeLa cells treated with 1 µM Dexamethasone (Dex) for 6h.
IP was performed with 4.0 µg rabbit anti-GR antibody (H-300, Santa Cruz).
Recommendations for product and protocol optimization:
- Step A2
Spin cells for 5 min instead of 10 min at 500 g at 4°C (not RT) for saving time and to avoid aberrant DNA interactions that might occur if time between harvest and cross linking is too long.
- Step B8
Using a sonication device with a sounding rod with a total lysate volume of 100 uL (3 ChIP assays) is difficult to handle. As a consequence, you easily lose much sample by dispersion. Better incubate cell pellet in the indicated 100 uL buffer D/PI mix for 5 min on ice and add 1X ChIP buffer (containing PI) to a volume of 350 uL. After sonication, add 580 uL 1X ChIP buffer (incl. PI) to the lysate (step D2). From this step the total volume for each sample will be as indicated in the manual. Sonication of bigger sample volume will not compromise the outcome of your experiment!
- Step B11
Spin sonicated lysates for 10 min instead of 5 min at 14,000 g at 4°C.
General remark:
Switching temperatures from spinning to RT at step A2 and A3 does not change the experimental results. Better keep 4°C constantly.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
MR. Christian Marx
Verified customer
提交于 Jan 08 2014