l*****i
发帖数: 1163 | 1 请问有人用过3xFlag做CHIP－Seq么？可以给个Protocol么（未经许可，不会传阅）？
多谢 | M********r
发帖数: 142 | 2 pls refer http://www.nature.com/ncb/journal/v15/n4/full/ncb2702.html?WT.ec_id=NCB-201304
most important is ab.
Yi Zhang used F7452 sigma. nobody won't trust yi zhang ..
ChIP-seq sample preparation.
For Kdm2b ChIP, Kdm2b Flag-tag knock-in mESCs were fixed with 2 mM
ethylene glycol bis(succinimidylsuccinate) (Thermo Scientific) for 1 h
, followed by 10 min in 1% formaldehyde and 5 min in 0.125
;M glycine to sequence the reaction. Cells were lysed in 1% SDS, 10 mM
EDTA and 50 mM Tris–HCl (pH 8.0) and the DNA was fragmented to
approximately 200–400 bp by sonification (Branson Sonifier 450).
Immunoprecipitation was performed with 5 μg rabbit polyclonal anti-
Flag (F7425, Sigma), 5 μl rabbit polyclonal Ring1b antibody (5694,
Cell Signaling Technology) and 5 μl rabbit polyclonal Ezh2 antibody (
5246, Cell Signaling Technology) overnight at 4 °C. Antibody-bound
protein was isolated by protein G plus/protein A agarose beads (EMD
Millipore), washed, eluted and reverse cross-linked. DNA was extracted by
phenol/chloroform and precipitated.
【在 l*****i 的大作中提到】
: 请问有人用过3xFlag做CHIP－Seq么？可以给个Protocol么（未经许可，不会传阅）？
: 多谢
| l*****i
发帖数: 1163 | 3 Many thanks. Baozi Sent.
Does anyone know whether the M2 antibody works for Flag CHIP?
;h
8201;
mM
to
【在 M********r 的大作中提到】
: pls refer http://www.nature.com/ncb/journal/v15/n4/full/ncb2702.html?WT.ec_id=NCB-201304
: most important is ab.
: Yi Zhang used F7452 sigma. nobody won't trust yi zhang ..
: ChIP-seq sample preparation.
: For Kdm2b ChIP, Kdm2b Flag-tag knock-in mESCs were fixed with 2 mM
: ethylene glycol bis(succinimidylsuccinate) (Thermo Scientific) for 1 h
: , followed by 10 min in 1% formaldehyde and 5 min in 0.125
: ;M glycine to sequence the reaction. Cells were lysed in 1% SDS, 10 mM
: EDTA and 50 mM Tris–HCl (pH 8.0) and the DNA was fragmented to
: approximately 200–400 bp by sonification (Branson Sonifier 450).
| M********r
发帖数: 142 | 4 no no no never M2 beads.
I compared and it never works.
2 reasons
1. ChIP buffer contains SDS and SDS kills M2 beads (refer to M2 beads
handbook)
2. you never know how much antibody you used. 10 ul of M2 beads perhaps
contain hundreds ug of ab. and saturate your chromatin. you need titration,
but impossible.
you may want to try M2 antibody which is a monoclonal antibody and the same
as the one conjugated with M2 beads.
You could buy M2 antibody and F ... polyclonal antibody and use both. you
could do ChIP-qPCR and compare.
but I suggest you use the one Yi Zhang published.
need more baozi.
【在 l*****i 的大作中提到】
: Many thanks. Baozi Sent.
: Does anyone know whether the M2 antibody works for Flag CHIP?
:
: ;h
: 8201;
: mM
: to
| l*****i
发帖数: 1163 | 5 Very helpful. 2 more baozi sent
titration,
same
【在 M********r 的大作中提到】
: no no no never M2 beads.
: I compared and it never works.
: 2 reasons
: 1. ChIP buffer contains SDS and SDS kills M2 beads (refer to M2 beads
: handbook)
: 2. you never know how much antibody you used. 10 ul of M2 beads perhaps
: contain hundreds ug of ab. and saturate your chromatin. you need titration,
: but impossible.
: you may want to try M2 antibody which is a monoclonal antibody and the same
: as the one conjugated with M2 beads.
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