Pluripotency Transcription Factor Antibody Kit
cat.: HAK21089
Product Type: Antibody Sampler Kit
Species reactivity: Human, Mouse
Applications: WB, IHC-P, IF-Tissue, IF-Cell, ChIP
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: PBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: Q9H9S0 Human | Q80Z64 Mouse | Q01860 Human | P20263 Mouse | P48431 Human | P48432 Mouse
Entrez Gene: 499593 Rat
Images
HAK21089_1.jpg Fig1: Western blot analysis of Nanog on different lysates with Rabbit anti-Nanog antibody (ET1610-2) at 1/2,000 dilution.

Lane 1: NCCIT cell lysate
Lane 2: HeLa cell lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 35 kDa
Observed band size: 40 kDa

Exposure time: 3 minutes;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1610-2) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21089_2.jpg Fig2: ICC staining of Nanog in F9 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1610-2, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
HAK21089_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human seminoma tissue with Rabbit anti-Nanog antibody (ET1610-2) at 1/800 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1610-2) at 1/800 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21089_4.jpg Fig4: Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells with Nanog (ET1610-2) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
HAK21089_5.jpg Fig5: Western blot analysis of Oct4 on different lysates with Rabbit anti-Oct4 antibody (ET1612-20) at 1/1,000 dilution.

Lane 1: F9 cell lysate
Lane 2: NCCIT cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 39 kDa
Observed band size: 45 kDa

Exposure time: 1 minute 59 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1612-20) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21089_6.jpg Fig6: Immunocytochemistry analysis of NCCIT cells labeling Oct4 with Rabbit anti-Oct4 antibody (ET1612-20) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Oct4 antibody (ET1612-20) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HAK21089_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded human seminoma tissue with Rabbit anti-Oct4 antibody (ET1612-20) at 1/4,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-20) at 1/4,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21089_8.jpg Fig8: Oct4 was immunoprecipitated in 0.2mg NCCIT cell lysate with ET1612-20 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using ET1612-20 at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: NCCIT cell lysate (input)
Lane 2: ET1612-20 IP in NCCIT cell lysate
Lane 3: Rabbit IgG instead of ET1612-20 in NCCIT cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 1 minute 40 seconds
HAK21089_9.jpg Fig9: Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells with Oct4 (ET1612-20) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
HAK21089_10.jpg Fig10: Western blot analysis of SOX2 on different lysates with Rabbit anti-SOX2 antibody (HA721155) at 1/2,000 dilution.

Lane 1: NCCIT cell lysate
Lane 2: F9 cell lysate
Lane 3: HeLa cell lysate (negative)

Predicted band size: 34 kDa
Observed band size: 34 kDa

Exposure time: 15 seconds;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721155) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21089_11.jpg Fig11: Immunocytochemistry analysis of F9 cells labeling SOX2 with Rabbit anti-SOX2 antibody (HA721155) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-SOX2 antibody (HA721155) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HAK21089_12.jpg Fig12: Immunohistochemical analysis of paraffin-embedded human glioma tissue with Rabbit anti-SOX2 antibody (HA721155) at 1/4,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA721155) at 1/4,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HAK21089_13.jpg Fig13: Immunofluorescence analysis of paraffin-embedded E14.5 mouse embryonic brain tissue labeling SOX2 with Rabbit anti-SOX2 antibody (HA721155) at 1/500 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (HA721155, green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).
HAK21089_14.jpg Fig14: Immunofluorescence analysis of frozen E14.5 mouse embryonic brain tissue with Rabbit anti-SOX2 antibody (HA721155) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (HA721155, green) at 1/1,000 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).
HAK21089_15.jpg Fig15: Chromatin immunoprecipitations were performed with cross-linked chromatin from NCCIT cells with SOX2 (HA721155) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
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