Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

Intracellular flow cytometric analysis of4% 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• ChIP

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ChIP - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• IP

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Immunoprecipitation - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

All lanes:

Immunoprecipitation - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (ab220788)

Predicted band size: 38 kDa

Observed band size: 45 kDa

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• ChIP-seq

Lab

ChIP-sequencing - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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 ab220788. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. ChIP-Seq validation performed with ChIP-Kit Transcription Factors ChIP-Seq (ab270813). Additional screenshots of mapped reads can be downloaded here.

• ChIP-seq

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ChIP-sequencing - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

• WB

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Western blot - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

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.

All lanes:

Western blot - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (ab220788) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 3:

HCT 116 (human colorectal carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 5:

C2C12 (mouse myoblast cell line) whole cell lysate at 20 µg

Lane 6:

Mouse testis lysate at 20 µg

Lane 7:

Rat testis lysate at 20 µg

Lane 8:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 38 kDa

Observed band size: 35 kDa,45 kDa

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• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 3x10^5 HeLa cells and 3µg of ab220788 [EPR21954]. The resulting DNA was sequenced on the NovaSeq 6000 (PE150). Additional screenshots of mapped reads can be downloaded here.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-TATA binding protein TBP antibody [EPR21954] - Loading Control and ChIP Grade (AB220788)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 3 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 3µg of ab220788 [EPR21954]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The ChIP data was conducted on chromatin 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 ab220788 with ChIP-Kit Transcription Factors ChIP-Seq (ab270813). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.