DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #14793

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

293 T cells were transiently transfected with GLI2-Flag and fixed with 1% formaldehyde for 15 min and quenched with 125 mM glycine for 5 min. Cells were washed with cold PBS and collected as 107 cell pellets. Cells were resuspended in 500 μL cold L1 buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.1% NP-40, 10% glycerol, 1 mM PMSF, 1x Pierce Protease Inhibitor) on ice for 5 min, centrifuged and resuspended in 450 μL cold SDS buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, 1x Pierce Protease Inhibitor). Chromatin was sheared for 10 cycles using the Bioruptor Pico to obtain fragments of 200–500 base pairs, and diluted 1:10 in cold ChIP buffer (0.5% NP-40, 5 mM EDTA, 200 mM NaCl, 50 μM Tris pH 8.0, 1 mM PMSF). Diluted chromatin was pre-cleared with 100 μL washed Protein A Dynabeads (Invitrogen) at 4°C for 1 hr then immunoprecipitated with Flag antibody (CST, 14793) or negative control Rabbit IgG (CST, 2729) at 4°C overnight. Immunocomplexes were recovered using 50 μL washed Protein A Dynabeads at 4°C for 2 hr. Beads were washed two times in following buffers: 800 μL Wash buffer (0.1% SDS, 1% NP-40, 2 mM EDTA, 500 mM NaCl, 20 mM Tris pH 8.0, 1 mM PMSF), LiCl buffer (0.1% SDS, 1% NP-40, 2 mM EDTA, 0.5M LiCl, 20 mM Tris pH 8.0, 1 mM PMSF) then TE buffer (1 mM EDTA, 10 mM Tris pH 8.0) for 5 min each all on ice. Complexes were eluted by resuspending beads in 100 μL 2% SDS in TE buffer then de-crosslinked overnight with 5 μL NaCl at 65°C. Recovered DNA was PCR purified and analyzed using qPCR. Primers were used to amplify a region that contains a GLI binding site in the SPP1 promoter (−2324 to −2316) as reported in Kijewska et al. (2017). Primers targeting the ACTB promoter served as a negative control while primers targeting a known GLI-binding site in the BCL2 promoter (−957 to −949) served as a positive control (Regl et al., 2004).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing ChIP Anti-bodies FLAG using DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #14793 from Cell Signaling Technology.

Manufacturer protocol

Download the product protocol from Cell Signaling Technology for DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #14793 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing ChIP Anti-bodies FLAG using DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #14793 from Cell Signaling Technology. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.