- 44-298G - Invitrogen Antibodies CREB1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Phospho-CREB pSer133 Antibody was done on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-CREB pSer133 Antibody (Product # 44-298G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of CREB (pS133) showing staining in the nucleus of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a CREB (pS133) polyclonal antibody (Product # 44-298G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of CREB (pS133) showing staining in the nucleus of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a CREB (pS133) polyclonal antibody (Product # 44-298G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of CREB [PS133] was done on A549 cells treated with EGF (200ng/mL, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with CREB [PS133] Rabbit Polyclonal Antibody (44298G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP- qPCR analysis of CREB pSer133 was performed with 10 µL of the Phospho-CREB pSer133 Rabbit polyclonal antibody (Product # 44-298G) on sheared chromatin from 2 million HeLa cells treated with 50 ng/mL of TNF Alpha for one hour using the MAGnify™ Chromatin Immunoprecipitation System (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA from each ChIP sample was analyzed by StepOnePlus™ Real-Time PCR System (Product # 4376600) with primers for the promoter of active CREB, IkB gene, used as positive control target, and the SAT2 used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

44-298G