Anti-CITED2 antibody [EPR27399-62]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HT-1080 (human fibrosarcoma epithelial cell) cells labelling CITED2 with ab314757 at 1/50 (10.34 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in HT-1080 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2ug/ml) dilution.

• IP

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Immunoprecipitation - Anti-CITED2 antibody [EPR27399-62] (AB314757)

CITED2 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab314757 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314757 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug Lane 2 : ab314757 IP in K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314757 in K-562 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CITED2 antibody [EPR27399-62] (ab314757) at 1/30 dilution

All lanes:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3min

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling CITED2 with ab314757 at 1/500 (1.034 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat colon. The section was incubated with ab314757 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling CITED2 with ab314757 at 1/500 (1.034 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse colon. The section was incubated with ab314757 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Immunohistochemical analysis of paraffin-embedded mouse pancreatic cancer tissue labeling CITED2 with ab314757 at 1/500 (1.034 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse pancreatic cancer. The section was incubated with ab314757 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling CITED2 with ab314757 at 1/500 (1.034 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat cerebrum. The section was incubated with ab314757 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling CITED2 with ab314757 at 1/500 (1.034 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse cerebrum. The section was incubated with ab314757 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-CITED2 antibody [EPR27399-62] (AB314757)

CITED2 was immunoprecipitated from 0.35 mg MEF (mouse embryo fibroblast) treated with 5 μM MG-132 for 6 hours whole cell lysate with ab314757 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314757 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : MEF (mouse embryo fibroblast) treated with 5 μM MG-132 for 6 hours whole cell lysate Lane 2 : ab314757 IP in MEF (mouse embryo fibroblast) treated with 5 μM MG-132 for 6 hours whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314757 in MEF treated with 5 μM MG-132 for 6 hours whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CITED2 antibody [EPR27399-62] (ab314757) at 1/30 dilution

All lanes:

MEF (mouse embryo fibroblast) treated with 5 μM MG-132 for 6 hours whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3min

• WB

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Western blot - Anti-CITED2 antibody [EPR27399-62] (AB314757)

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

The expression profile observed is consistent with what has been described in the literature (PMID : 17906695).

Lysates were freshly made and used immediately to minimize protein degradation.

In Western Blot, Anti HIF-1 alpha antibody [EPR16897-101] (ab179483) is applied at 1/1000 dilution to prove the DFO induction is successful.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CITED2 antibody [EPR27399-62] (ab314757) at 1/1000 dilution

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Lane 3:

HT-1080 treated with 0.5 mM DFO for 24 hours, then added 10uM MG-132 for 4 hours, whole cell lysate at 20 µg

Lane 4:

HT-1080 treated with 10uM MG-132 for 4 hours, 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

Observed band size: 30 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lane 1-3 : 48 seconds Lane 4 : 37 seconds

All lanes:

Western blot - Anti-CITED2 antibody [EPR27399-62] (ab314757) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

K-562 (human chronic myelogenous leukemia lymphoblast) 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

Observed band size: 30 kDa

false

• WB

Supplier Data

Western blot - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression profile observed is consistent with what has been described in the literature (PMID : 17906695). The band beneath the target band (30 kDa) is expected to be degradation products. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. Exposure time : Lane 1, 2 : 15 seconds Lane 3, 4 : 103 seconds

All lanes:

Western blot - Anti-CITED2 antibody [EPR27399-62] (ab314757) at 1/1000 dilution

Lane 1:

Untreated MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 2:

MEF treated with 5 uM MG-132 for 6 hours, whole cell lysate at 20 µg

Lane 3:

Untreated PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate at 20 µg

Lane 4:

PC-12 treated with 20 uM MG-132 for 5 hours, 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

Observed band size: 30 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used immediately to minimize protein degradation.

All lanes:

Western blot - Anti-CITED2 antibody [EPR27399-62] (ab314757) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue 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

Observed band size: 30 kDa

false

Exposure time: 3min

• Dot

Supplier Data

Dot Blot - Anti-CITED2 antibody [EPR27399-62] (AB314757)

Dot blot analysis of CITED2 using ab314757 at 1 : 1000 (0.517 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Exposure time : 48 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST