Anti-CREB (phospho S133) antibody [E113]

19 product images

• 

  Immunoprecipitation - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  ab32096 at 1/100 dilution immunoprecipitating CREB (phospho S133) in HeLa (human cervix adenocarcinoma) treated with 25ug/mL anisomycin for 30 minutes, whole cell lysate, observed at 40 kDa (lanes 1 and 2).
  

  Lane 1 (input): HeLa treated with 25ug/mL anisomycin for 30 minutes. Whole cell lysate, 10μg.

  Lane 2 (+): ab32096 + HeLa treated with 25ug/mL anisomycin for 30 minutes. Whole cell lysate.

  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32096 in treated with 25ug/mL anisomycin for 30 minutes. Whole cell lysate

  For western blotting, ab32096 at 1/200 dilution followed by VeriBlot for IP Detection Reagent (HRP) ab131366 VeriBlot for IP Detection Reagent (HRP) at 1/1000 for detection.

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

  All lanes: Immunoprecipitation - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Predicted band size: 36 kDa

  Exposure time: 5s

• 

  Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096)

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

  All lanes: Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096) at 1/500 dilution

  Lane 1: Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates at 15 µg

  Lane 2: HeLa whole cell lysates treated with anisomycin at 25ug/ml for 30 minutes at 15 µg

  Lane 3: HeLa whole cell lysates treated with anisomycin at 25ug/ml for 30 minutes, then the membrane was incubated with phosphatase at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 36 kDa

  Observed band size: 40 kDa

  Exposure time: 15s

• 

  Immunoprecipitation - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  ab32096 (purified) at 1/50 immunoprecipitating CREB (phospho S133) in HeLa whole cell lysate. 10 ug of cell lysate was present in the input. For western blotting, a HRP-conjugated Veriblot for IP Detection Reagent (VeriBlot for IP Detection Reagent (HRP) ab131366) (1/1,500) was used for detection. A rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) was used intead of Anti-Rab11A antibody [EPR7587(B)] ab128913 as a negative control (Lane 3).
  

  Blocking buffer and concentration: 5% NFDM/TBST.

  Diluting buffer and concentration: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Predicted band size: 36 kDa

• 

  Flow Cytometry (Intracellular) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Intracellular Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling CREB with purified ab32096 at 1/70 dilution (10μg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.
  

• 

  Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Blocking and diluting buffer: 5% NFDM/TBST
  

  This antibody can`t detect signal in mouse and rat brain related tissues.

  All lanes: Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096) at 1/200 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) treated with 250ng/ml anisomycin for 30 minutes whole cell lysates at 15 µg

  Lane 2: Mouse brain lysates at 15 µg

  Lane 3: Rat brain lysates at 15 µg

  Lane 4: Rat cerebellum lysate at 15 µg

  Lane 5: Mouse hippocampus lysate at 15 µg

  Lane 6: Rat hippocampus lysate at 15 µg

  Lane 7: Rat cerebral cortex lysate at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 36 kDa

  Exposure time: 3s

• 

  Immunocytochemistry/ Immunofluorescence - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunocytochemistry/Immunofluorescence analysis of LP treated and untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) labelling CREB with purified ab32096 at 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain at a dilution of 1/200.
  

  Confocal image showing nuclear staining on HeLa cells .The signal decreased after Lambda Protein Phosphatase treatment ( 31°C,2h).

• 

  Functional Studies - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  SK-N-SH cells were incubated at 37°C for 30 minutes with vehicle control (0 μM) and different concentrations of (R,S)-CHPG ((R,S)-CHPG, mGlu5 agonist ab120039). Increased expression of CREB (phospho S133) in SK-N-SH cells correlates with an increase in (R,S)-CHPG concentration, as described in literature.

  Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with unpurified ab32096 at 1/500 dilution and Anti-CREB antibody [E306] ab32515 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051 ) at 1/10000 dilution and visualised using ECL development solution.

• 

  Functional Studies - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  SK-N-SH cells were incubated at 37°C for 30 minutes with vehicle control (0 μM) and different concentrations of (S)-3,5-DHPG ((S)-3,5-DHPG, group I mGlu agonist ab120007). Increased expression of CREB (phospho S133) in SK-N-SH cells correlates with an increase in (S)-3,5-DHPGconcentration, as described in literature.

  Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with unpurified ab32096 at 1/500 dilution and Anti-CREB antibody [E306] ab32515 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/10000 dilution and visualised using ECL development solution.

• 

  Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096)

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

  Lane 1: Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096) at 1/5000 dilution

  Lane 2: purified at 1/5000 dilution

  Lane 1: Untreated C6 cell lysate at 10 µg

  Lane 2: C6 treated with Calyculin A cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/1000 dilution

  Predicted band size: 36 kDa

• 

  Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096)

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

  All lanes: Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096) at 1/5000 dilution

  Lane 1: Untreated NIH/3T3 cell lysate at 10 µg

  Lane 2: NIH/3T3 treated with Calyculin A at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/1000 dilution

  Predicted band size: 36 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunocytochemistry/Immunofluorescence analysis of A431(human epidermoid carcinoma) cells +/- AP 37°C 1h labelling CREB with purified ab32096 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

  Control 1: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• 

  Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096)

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

  All lanes: Western blot - Anti-CREB (phospho S133) antibody [E113] (ab32096) at 1/5000 dilution

  Lane 1: Untreated HeLa cell lysate at 10 µg

  Lane 2: HeLa treated with Calyculin A cell lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/1000 dilution

  Predicted band size: 36 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling CREB with purified ab32096 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling CREB with unpurified ab32096 at 1/250.

  Panel A: Cells are untreated.

  Panel B: Cells are treated with Phosphatase.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid gland adenocarcinoma tissue labelling CREB with unpurified ab32096 at 1/250 dilution.

  Panel A: Cells are untreated.
  Panel B: Cells are treated with Phosphatase.

• 

  This image is courtesy of a customer review submitted by Akiko Shingo.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat hippocampus tissue labelling CREB with unpurified ab32096 at 1/100 dilution. Sections were subjected to antigen retrieval by autoclave prior to blocking with 8% milk for 30 minutes at 37°C. The primary antibody was diluted 1/100 with DAKO antibody diluent and incubated with the sample for 18 hours at 4°C. An LSAB-labeled Streptavidin-Biotin conjugated Goat polyclonal antibody was used undiluted as the secondary antibody.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling CREB with ab32096 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB ab209101. Nuclear staining on rat cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab32096 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 Rabbit specific IHC polymer detection kit HRP/DAB ab209101.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling CREB with ab32096 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB ab209101. Nuclear staining on mouse cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab32096 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 Rabbit specific IHC polymer detection kit HRP/DAB ab209101.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREB (phospho S133) antibody [E113] (ab32096)

  Immunohistochemical analysis of paraffin-embedded Human astrocytoma tissue labeling CREB with ab32096 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB ab209101. Nuclear staining on human astrocytoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab32096 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 Rabbit specific IHC polymer detection kit HRP/DAB ab209101.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.