All lanes : 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
Lane 2 : HeLa whole cell lysates treated with anisomycin at 25ug/ml for 30 minutes
Lane 3 : HeLa whole cell lysates treated with anisomycin at 25ug/ml for 30 minutes, then the membrane was incubated with phosphatase

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 37 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

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

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. 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℃,2h).

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. 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.

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 Fluor^® 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.

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 (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 ab131366 VeriBlot for IP Detection Reagent (HRP) at 1/1000 for detection.

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

All lanes : 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
Lane 2 : Mouse brain lysates
Lane 3 : Rat brain lysates
Lane 4 : Rat cerebellum lysate
Lane 5 : Mouse hippocampus lysate
Lane 6 : Rat hippocampus lysate
Lane 7 : Rat cerebral cortex lysate

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 37 kDa


Exposure time: 3 seconds

Blocking and diluting buffer: 5% NFDM/TBST

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

Immunocytochemistry/Immunofluorescence analysis of A431(human epidermoid carcinoma) cells +/- AP 37℃ 1h labelling CREB with purified ab32096 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. 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. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

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.

See Abreview

SK-N-SH cells were incubated at 37&degC for 30 minutes with vehicle control (0 &microM) and different concentrations of (S)-3,5-DHPG (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), 20&microg 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 ab32515 at 1 &microg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

 

Lane 1 : Anti-CREB (phospho S133) antibody [E113] (ab32096) at 1/5000 dilution (purified)
Lane 2 : purified at 1/5000 dilution

Lane 1 : Untreated C6 cell lysate
Lane 2 : C6 treated with Calyculin A cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 37 kDa

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

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

Lane 1 : Untreated NIH/3T3 cell lysate
Lane 2 : NIH/3T3 treated with Calyculin A

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 37 kDa

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

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

Lane 1 : Untreated HeLa cell lysate
Lane 2 : HeLa treated with Calyculin A cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 37 kDa

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

SK-N-SH cells were incubated at 37&degC for 30 minutes with vehicle control (0 &microM) and different concentrations of (R,S)-CHPG (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), 20&microg 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 ab32515 at 1 &microg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051 ) at 1/10000 dilution and visualised using ECL development solution.

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 (ab131366) (1/1,500) was used for detection. A rabbit monoclonal IgG (ab172730) was used intead of ab128913 as a negative control (Lane 3).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

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.

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.