Phospho-Creb (S133) Recombinant Rabbit Monoclonal Antibody [JB25-40]
cat.: ET7107-93
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P, IP, FC, IF-Tissue
Clonality: Monoclonal
Clone number: JB25-40
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 35 kDa
Isotype: IgG
Immunogen: Synthetic phospho-peptide corresponding to residues surrounding Ser133 of human Creb.
Positive control: HeLa treated with 25μg/mL anisomycin for 30 minutes whole cell lysate, SH-SY5Y, HUVEC, human spleen tissue, mouse colon tissue, human colon carcinoma tissue, human lymph nodes tissue, mouse large intestine tissue, HeLa.
Subcellular location: Nucleus.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
  FC
  IP
  IF-Tissue
1:1,000
1:50-1:100
1:50-1:1,000
1:50-1:100
Use at an assay dependent concentration.
1:200
Uniprot #: SwissProt: P16220 Human | Q01147 Mouse
Alternative names: Active transcription factor CREB cAMP response element binding protein 1 cAMP response element binding protein cAMP responsive element binding protein 1 cAMP-responsive element-binding protein 1 CREB CREB-1 CREB1 CREB1_HUMAN Cyclic AMP-responsive element-binding protein 1 MGC9284 OTTHUMP00000163864 OTTHUMP00000163865 OTTHUMP00000206660 OTTHUMP00000206662 OTTHUMP00000206667 Transactivator protein
Images
ET7107-93_1.jpg Fig1: Western blot analysis of Phospho-Creb (S133) on different lysates with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/1,000 dilution.

Lane 1: HeLa whole cell lysate
Lane 2: HeLa treated with 25μg/mL anisomycin for 30 minutes whole cell lysate

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 (ET7107-93) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.
ET7107-93_2.jpg Fig2: ICC staining of Phospho-Creb (S133) in SH-SY5Y 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 (ET7107-93, 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).
ET7107-93_3.jpg Fig3: ICC staining of Phospho-Creb (S133) in HUVEC 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 (ET7107-93, 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).
ET7107-93_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-Phospho-Creb (S133) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7107-93, 1/50) for 30 minutes 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.
ET7107-93_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded mouse colon tissue using anti-Phospho-Creb (S133) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7107-93, 1/50) for 30 minutes 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.
ET7107-93_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human lymph nodes tissue with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/400 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 (ET7107-93) at 1/400 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.
ET7107-93_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded mouse large intestine tissue with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/400 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 (ET7107-93) at 1/400 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.
ET7107-93_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/200 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 (ET7107-93) at 1/200 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.
ET7107-93_9.jpg Fig9: Flow cytometric analysis of Phospho-Creb (S133) was done on HUVEC cells. The cells were fixed, permeabilized and stained with the primary antibody (ET7107-93, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
ET7107-93_10.jpg Fig10: Immunocytochemistry analysis of HeLa cells treated with or without Lambda Protein Phosphatase for 1 hour labeling Phospho-Creb (S133) with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. 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.
ET7107-93_11.jpg Fig11: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/1,000 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 (ET7107-93) at 1/1,000 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.
ET7107-93_12.jpg Fig12: Immunohistochemical analysis of paraffin-embedded rat colon tissue with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/1,000 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 (ET7107-93) at 1/1,000 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.
ET7107-93_13.jpg Fig13: Immunocytochemistry analysis of NIH/3T3 cells labeling Phospho-Creb (S133) with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 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-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 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.
ET7107-93_14.jpg Fig14: Immunocytochemistry analysis of C6 cells labeling Phospho-Creb (S133) with Rabbit anti-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 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-Phospho-Creb (S133) antibody (ET7107-93) at 1/100 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.