Application
ChIP
Sample
Human Cell lysate - nuclear (LNCaP-1F5 Cell line)
Specification
LNCaP-1F5 Cell line
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Detection step
Real-time PCR
Positive control
PSA enhancer ~4 kb upstream of TSS
Negative control
Negative control region on Chromosome X not known to be bound by any nuclear receptors.
Other product details
Concentration
5 µl/µg chromatin
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: SDS, DOC, TritonX-100, EDTA, HEPES, NaCl
Additional data
Additional Notes
Checked the fold enrichment on PSA enhancer compared it with another commercial monoclonal antibody, which works great for direct ChIP assay but we didn't get good results form sequencing, so ordered the ChIP grade Abcam antibody, but this seems to be worse than the previous commercial one. The fold enrichment here refers to relative occupancy in presence of Ethanol as vehicle Control (Ctrl-GR), with Dexamethsone treated cells (DEX-GR), normalised to each of their own input.
The ChIP was done with the GR antibody (ab3579) for control and dexamethsone treated cells and also an IgG control was included to see if the enrichment is good but we are not able to see a ligand dependent difference.
This was really astonishing to find that the IgG was mich higher than the vehicle control, maybe it needs more optimisation in pre-clearing, or in the washing steps. But we failed with increasing the washes as the amount of IP DNA enriched was getting lower.
We even tried to make two lanes of Solexa sequencing with the DEX-treated samples IP'ed with GR antibody, but all the peaks were too weak to be significantly called as true binding sites after sequencing. When we run our control AR ChIP samples on sequencing in 2 lanes, we get around 15000 binding sites enriched at less than 2% FDR, but with same GR ChIP samples we get around 1000-1500 peaks and the FDR values computed are also too high.
One more thing i would like to point here is this might be completely because of this cell line and the same doesn't hold true for another ones, though we haven't tried ourselves.
The ChIP was done with the GR antibody (ab3579) for control and dexamethsone treated cells and also an IgG control was included to see if the enrichment is good but we are not able to see a ligand dependent difference.
This was really astonishing to find that the IgG was mich higher than the vehicle control, maybe it needs more optimisation in pre-clearing, or in the washing steps. But we failed with increasing the washes as the amount of IP DNA enriched was getting lower.
We even tried to make two lanes of Solexa sequencing with the DEX-treated samples IP'ed with GR antibody, but all the peaks were too weak to be significantly called as true binding sites after sequencing. When we run our control AR ChIP samples on sequencing in 2 lanes, we get around 15000 binding sites enriched at less than 2% FDR, but with same GR ChIP samples we get around 1000-1500 peaks and the FDR values computed are also too high.
One more thing i would like to point here is this might be completely because of this cell line and the same doesn't hold true for another ones, though we haven't tried ourselves.
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.
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提交于 Feb 02 2010