Anti-Nanog antibody [EPR20694] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Immunohistochemical analysis of paraffin-embedded adult mouse testis tissue labeling Nanog with ab214549 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Negative tissue : No staining on adult mouse testis is observed(PMID : 12787505; PMID : 15939376).

Counter stained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Intracellular flow cytometric analysis of 4 % paraformaldehyde-fixed, 90% methanol permeabilized F9 (mouse embryonic testicular cancer cell line, Right) and NIH/3T3 (mouse embryo fibroblast cell line, Left) cells labeling Nanog with ab214549 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : NIH/3T3 cell line.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Immunohistochemical analysis of paraffin-embedded mouse E14.5 testis tissue labeling Nanog with ab214549 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Mainly nuclear staining is observed in testis of mouse embryo E14.5 (PMID : 15939376; PMID : 12787505). 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 Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeablized F9 (mouse embryonic testicular cancer cell line) and NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Nanog with ab214549 at 1/100 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 F9 cell line. Negative control : NIH/3T3 (PMID : 17352742).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

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

• IP

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Immunoprecipitation - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Nanog was immunoprecipitated from 0.35 mg of F9 (mouse embryonic testicular cancer cell line) whole cell lysate with ab214549 at 1/1000 dilution. Western blot was performed from the immunoprecipitate using ab214549 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution

Lane 1 : F9 whole cell lysate 10 μg (input)

Lane 2 : ab214549 IP in F9 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab214549 in F9 whole cell lysate.

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

Exposure time : 15 seconds.

The multiple bands observed are consistent with the literature (PMID : 24936455).

All lanes:

Immunoprecipitation - Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549)

Predicted band size: 34 kDa

false

• ChIP

Unknown

ChIP - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Chromatin was prepared from F9 cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 5μg of ab214549 (red), and 20 μl of protein A/G sepharose beads slurry (10 μl of sepharose A beads + 10 μl of sepharose G beads). Then 5 μg of rabbit normal IgG was added to the control beads (grey). The immunoprecipitated DNA was quantified by real time PCR (SYBR green chemistry).

• WB

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Western blot - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Blocking/Dilution buffer : 5% NFDM/TBST.

The multiple bands observed are consistent with the literature (PMID : 24936455).

All lanes:

Western blot - Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549) at 1/1000 dilution

All lanes:

ES-D3 (mouse embryonic multipotent stem cell line) whole cell lysate at 10 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: 34 kDa

Observed band size: 29-42 kDa

true

Exposure time: 3min

• WB

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Western blot - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

Blocking/Dilution buffer : 5% NFDM/TBST.

The multiple bands observed are consistent with the literature (PMID : 24936455). Negative control : NIH/3T3 (PMID : 12787505).

All lanes:

Western blot - Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549) at 1/1000 dilution

Lane 1:

F9 (Mouse embryonic testicular cancer cell line) whole cell lysate at 20 µg

Lane 2:

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: 34 kDa

Observed band size: 29-42 kDa

true

Exposure time: 70s

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-Nanog antibody [EPR20694] - ChIP Grade (AB214549)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 F9 (Mouse embryonic testicular cancer cell line) cells and 5µg of ab214549 [EPR20694]. 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. 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.