Anti-CREB antibody [LB9]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB antibody [LB9] (AB178322)

Immunohistochemical analysis of deparaffinized Human tonsil tissue labeling CREB with ab178322 at 1/100 dilution followed by DAB staining.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CREB antibody [LB9] (AB178322)

Immunofluorescent analysis of untreated HeLa cells (formalin-fixed, 0.1% Triton X-100 permeabilized) labeling CREB with ab178322 at 1/400 dilution (green) followed with DyLight 488 goat anti-mouse IgG secondary antibody at 1/400 dilution. Nuclei (blue) were stained with Hoechst 33342 dye.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CREB antibody [LB9] (AB178322)

Immunofluorescent analysis of untreated HeLa cells (formalin-fixed, 0.1% Triton X-100 permeabilized) labeling CREB with ab178322 at 1/500 dilution (green) followed with DyLight 488 goat anti-mouse IgG secondary antibody at 1/400 dilution. Nuclei (blue) were stained with Hoechst 33342 dye.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB antibody [LB9] (AB178322)

Immunohistochemical analysis of deparaffinized Human bladder cancer (left) and rectal cancer (right) tissues labeling CREB with ab178322 at 1/200 dilution followed by DAB staining.

• IP

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Immunoprecipitation - Anti-CREB antibody [LB9] (AB178322)

Immunoprecipitation analysis of CREB was performed on untreated 293T cells. The antigen : antbody complex was formed by binding 500μg whole cell lysate with 2μg ab178322 and captured on 50μl Protein A/G Plus Agarose.

Lane 1 : 293T lysate

Lane 2 : IP of 293T lysate

WB detection used ab178322 at 1/1000 followed with a goat anti-mouse-HRP secondary antibody at 1/20,000.

All lanes:

Immunoprecipitation - Anti-CREB antibody [LB9] (ab178322)

Predicted band size: 36 kDa

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• WB

Lab

Western blot - Anti-CREB antibody [LB9] (AB178322)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : CREB knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : A431 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab178322 observed at 44 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab178322 was shown to recognize CREB when CREB knockout samples were used, along with additional cross-reactive bands. Wild-type and CREB knockout samples were subjected to SDS-PAGE. ab178322 and ab18058 (loading control to Vinculin) were diluted at 1/250 and 1/10,000 dilution respectively and incubated overnight at 4C. Blots were developed with IRDye® 800CW Goat anti-Rabbit IgG (H + L) and IRDye® 680 Goat anti-Mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CREB antibody [LB9] (ab178322)

Predicted band size: 36 kDa

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• WB

Lab

Western blot - Anti-CREB antibody [LB9] (AB178322)

Western blot : Anti-CREB1 antibody [LB9] (ab178322) staining at 1/500 dilution, shown in green; Rabbit anti-ACTN4 [EPR2533(2)] (ab108198) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab178322 was shown to bind specifically to CREB1. A band was observed at 40 kDa in wild-type A549 cell lysates with no signal observed at this size in CREB1 knockout cell line. To generate this image, wild-type and CREB1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CREB antibody [LB9] (ab178322) at 1/500 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

CREB1 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type A549 ab288558 cell lysate at 20 µg

Lane 4:

CREB1 knockout A549 <a href='/en-us/products/cell-lines/human-creb1-knockout-a549-cell-line-ab286720'>ab286720</a> cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Predicted band size: 36 kDa

Observed band size: 40 kDa

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• WB

Supplier Data

Western blot - Anti-CREB antibody [LB9] (AB178322)

All lanes:

Western blot - Anti-CREB antibody [LB9] (ab178322) at 1/500 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 30 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 30 µg

Lane 3:

K-562 (Human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate at 30 µg

Lane 4:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 30 µg

Lane 5:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 30 µg

Lane 6:

C2C12 whole cell lysate at 30 µg

Lane 7:

U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate at 30 µg

Lane 8:

A549 (Human lung carcinoma cell line) whole cell lysate at 30 µg

Lane 9:

MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate at 30 µg

Lane 10:

T98G whole cell lysate at 30 µg

Predicted band size: 36 kDa

Observed band size: 43 kDa

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• WB

Supplier Data

Western blot - Anti-CREB antibody [LB9] (AB178322)

All lanes:

Western blot - Anti-CREB antibody [LB9] (ab178322) at 1/500 dilution

Lane 1:

HeLa whole cell lysate at 25 µg

Lane 2:

293T whole cell lysate at 25 µg

Lane 3:

A431 whole cell lysate at 25 µg

Lane 4:

A549 whole cell lysate at 25 µg

Lane 5:

MEF whole cell lysate at 25 µg

Lane 6:

MCF7 whole cell lysate at 25 µg

Lane 7:

U2OS whole cell lysate at 25 µg

Lane 8:

K562 whole cell lysate at 25 µg

Lane 9:

COS7 whole cell lysate at 25 µg

Lane 10:

3T3L1 whole cell lysate at 25 µg

Lane 11:

C2C12 whole cell lysate at 25 µg

Lane 12:

NRK whole cell lysate at 25 µg

Secondary

All lanes:

goat anti-mouse-HRP at 1/20000 dilution

Predicted band size: 36 kDa

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