Bcl-11B (D6F1) XP® Rabbit mAb #12120

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.	-For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.	-Once in solution, store 1M DTT at -20°C.

Upstream tips
-It is highly critical that the chromatin is of appropriate size and concentration.
Protocol tips
-For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. -It is important to keep the tissue cold to avoid protein degradation. -Use fresh formaldehyde that is not past the manufacturer's expiration date.
Downstream tips
-Once in solution, store 1M DTT at -20°C.

Publication protocol

Treg cells were isolated from the spleens and peLNs of naïve mice and enriched using the EasySep Mouse CD4+ CD25+ Regulatory T Cell Isolation Kit II (#18783, STEMCELL). Human Treg cells were isolated from PBMCs by sorting CD4+ CD25+ CD127− cells. Bcl11b ChIP-seq was performed on Treg cells using the SimpleChIP Enzymatic Chromatin IP Kit from Cell Signaling Technology (CST #9003), following their recommended protocol up to the library preparation with the following alterations: (i) 107 cells were used in place of 4 × 107, (ii) formaldehyde fixation was performed in 1 ml, (iii) buffer A and B steps were all performed in 1 ml, (iv) cells were sheared in ChIP buffer to an average of 200 to 1000 base pairs using a Diagenode Bioruptor Pico (B01060002), (v) a cocktail of three anti-Bcl11b antibodies were used (4 μg of ab18465, 4 μg of CST #12120, and 4 μg of Bethyl A300-385 per ChIP), and (vi) final DNA purification was performed by phenol:chloroform:isoamyl alcohol extraction and MaXtract high-density columns (#129046, Qiagen). Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (#E7645S, New England Biolabs) and sequenced as SE75 on a NextSeq500.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for Bcl-11B (D6F1) XP® Rabbit mAb #12120 below.

Download manufacturer protocol

Videos

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