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).

All lanes : Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549) at 1/1000 dilution

Lane 1 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
Lane 2 : 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

Developed using the ECL technique.

Predicted band size: 34 kDa
Observed band size: 29-42 kDa why is the actual band size different from the predicted?


Exposure time: 70 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

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

Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549) at 1/1000 dilution + ES-D3 (mouse embryonic multipotent stem cell line) whole cell lysate at 10 µg

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

Developed using the ECL technique.

Predicted band size: 34 kDa
Observed band size: 29-42 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.

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.

 

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.

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.

 

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).