Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. The ChIP was performed with 25 µg of chromatin, 5 µg of ab220788 (red), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).Primers and probes are located in the first kb of the transcribed region.

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-TATA binding protein TBP antibody [EPR21954] - ChIP Grade (ab220788). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.

Additional screenshots of mapped reads can be downloaded here.

All lanes : Anti-TATA binding protein TBP antibody [EPR21954] - ChIP Grade (ab220788) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : HCT 116 (human colorectal carcinoma cell line) whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 5 : C2C12 (mouse myoblast cell line) whole cell lysate
Lane 6 : Mouse testis lysate
Lane 7 : Rat testis lysate
Lane 8 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 38 kDa
Observed band size: 35,45 kDa why is the actual band size different from the predicted?

Exposure times : Lanes 1-7: 15 seconds, Lane 8: 3 minutes.

Blocking and dilution buffer: 5% NFDM/TBST.

Human TBP migrates with an approximate molecular mass of 45 kDa on SDS-PAGE (PMID:1458534; PMID: 1907890) the molecular mass observed is lower in mouse than in human samples due to a shorter sequence.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling TATA binding protein TBP with ab220788 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in mouse testis (PMID: 17570761; PMID: 11861477) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling TATA binding protein TBP with ab220788 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in rat testis (PMID: 17570761; PMID: 11861477) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

 

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling TATA binding protein TBP with ab220788 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in human bladder cancer (PMID: 17570761; PMID: 11861477) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

 

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling TATA binding protein TBP with ab220788 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in human testis (PMID: 17570761; PMID: 11861477) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

 

TATA binding protein TBP was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab220788 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220788 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg (Input).
Lane 2: ab220788 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab220788 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling TATA binding protein TBP with ab220788 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells incubated with secondary antibody only) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma cell line) cells labeling TATA binding protein TBP with ab220788 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HCT 116 cells. The nuclear counter stain is DAPI (blue). 

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling TATA binding protein TBP with ab220788 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HeLa cells. The nuclear counter stain is DAPI (blue). 

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.