重组
RabMAb

Anti-CREB (phospho S133)抗体[E113] (ab32096)

概述

  • 产品名称
    Anti-CREB (phospho S133)抗体[E113]
    参阅全部 CREB 一抗
  • 描述
    兔单克隆抗体[E113] to CREB (phospho S133)
  • 特异性
    This antibody is specific for CREB phosphorylated on Serine 133. The immunogen of the antibody shares 94%homology with CREM (S136) and 86% homology with ATF1 (pS63). No experiment has been performed to verify the possible cross-reactivity.
  • 经测试应用
    适用于: WB, IHC-P, IP, Flow Cyt, Dot blot, ICC/IFmore details
  • 种属反应性
    与反应: Mouse, Rat, Human
    预测可用于: Chicken, Cow, Zebrafish
  • 免疫原

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human CREB aa 100-200.
    Database link: P16220
    (Peptide available as ab182754)

  • 阳性对照
    • WB: A431 cell lysate. IF: A431 cells. IHC-P: Thyroid gland adenocarcinoma. IP: HeLa treated with 25ug/mL anisomycin for 30 minutes.
  • 常规说明

    This product is a recombinant rabbit monoclonal antibody.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

性能

应用

Our Abpromise guarantee covers the use of ab32096 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
WB 1/5000. Predicted molecular weight: 37 kDa.Can be blocked with CREB (phospho S133) peptide (ab182754).

For unpurified use at 1/500.

IHC-P 1/100 - 1/250.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

IP 1/40 - 1/100.

 

Flow Cyt Use at an assay dependent concentration.
Dot blot 1/1000.
ICC/IF 1/100 - 1/250.

靶标

  • 功能
    This protein binds the cAMP response element (CRE), a sequence present in many viral and cellular promoters. CREB stimulates transcription on binding to the CRE. Transcription activation is enhanced by the TORC coactivators which act independently of Ser-133 phosphorylation. Implicated in synchronization of circadian rhythmicity.
  • 疾病相关
    Defects in CREB1 may be a cause of angiomatoid fibrous histiocytoma (AFH) [MIM:612160]. A distinct variant of malignant fibrous histiocytoma that typically occurs in children and adolescents and is manifest by nodular subcutaneous growth. Characteristic microscopic features include lobulated sheets of histiocyte-like cells intimately associated with areas of hemorrhage and cystic pseudovascular spaces, as well as a striking cuffing of inflammatory cells, mimicking a lymph node metastasis. Note=A chromosomal aberration involving CREB1 is found in a patient with angiomatoid fibrous histiocytoma. Translocation t(2;22)(q33;q12) with CREB1 generates a EWSR1/CREB1 fusion gene that is most common genetic abnormality in this tumor type.
  • 序列相似性
    Belongs to the bZIP family.
    Contains 1 bZIP domain.
    Contains 1 KID (kinase-inducible) domain.
  • 翻译后修饰
    Stimulated by phosphorylation. Phosphorylation of both Ser-133 and Ser-142 in the SCN regulates the activity of CREB and participates in circadian rhythm generation. Phosphorylation of Ser-133 allows CREBBP binding (By similarity). Phosphorylated upon DNA damage, probably by ATM or ATR.
    Sumoylated by SUMO1. Sumoylation on Lys-304, but not on Lys-285, is required for nuclear localization of this protein. Sumoylation is enhanced under hypoxia, promoting nuclear localization and stabilization.
  • 细胞定位
    Nucleus.
  • Information by UniProt
  • 数据库链接
  • 别名
    • Active transcription factor CREB antibody
    • cAMP response element binding protein 1 antibody
    • cAMP response element binding protein antibody
    • cAMP responsive element binding protein 1 antibody
    • cAMP-responsive element-binding protein 1 antibody
    • CREB antibody
    • CREB-1 antibody
    • CREB1 antibody
    • CREB1_HUMAN antibody
    • Cyclic AMP-responsive element-binding protein 1 antibody
    • MGC9284 antibody
    • OTTHUMP00000163864 antibody
    • OTTHUMP00000163865 antibody
    • OTTHUMP00000206660 antibody
    • OTTHUMP00000206662 antibody
    • OTTHUMP00000206667 antibody
    • Transactivator protein antibody
    see all

图片

  • 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
    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

  • 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.
  • 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
    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 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).

  • 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 (HRP) at 1/1000 as the secondary antibody.

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

  • 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



  • Predicted band size : 37 kDa

    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
    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
    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
    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.


  • Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 37 kDa

    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.

  • 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).

  • 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 secondary antibody (ab131366) (1/1,500) was used as the secondary antibody. 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.

  • Dot blot analysis of CREB (pS133) phospho peptide (Lane 1) and CREB non-phospho peptide (Lane 2) using ab32096 at 1/1000 dilution followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution.

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

    Exposure time: 3 minutes.

文献

This product has been referenced in:
  • Liu Y  et al. Dibutyryl-cAMP attenuates pulmonary fibrosis by blocking myofibroblast differentiation via PKA/CREB/CBP signaling in rats with silicosis. Respir Res 18:38 (2017). WB, IHC-P ; Rat . Read more (PubMed: 28222740) »
  • Sarowar T  et al. Object Phobia and Altered RhoA Signaling in Amygdala of Mice Lacking RICH2. Front Mol Neurosci 10:180 (2017). WB ; Mouse . Read more (PubMed: 28642683) »

See all 26 Publications for this product

客户评价及客户问答

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse pancreatic cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
mouse pancreatic cells
Blocking step
odyssey blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Nov 01 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Hela)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
Hela
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Dr. Lin Zhu

Verified customer

提交于 Sep 04 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (BRAIN)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
BRAIN
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Username

Kyungjoo Seong

Verified customer

提交于 Jun 20 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Adult brain including cerebellum)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mM Citrate buffer pH 6
Permeabilization
No
Specification
Adult brain including cerebellum
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Fixative
Paraformaldehyde
Username

Dr. Estela Munoz

Verified customer

提交于 Dec 17 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Adult Cerebellum)
Gel Running Conditions
Reduced Denaturing (12.5% SDS-PAGE Gel)
Loading amount
30 µg
Specification
Adult Cerebellum
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: RT°C
Username

Dr. Estela Munoz

Verified customer

提交于 Dec 17 2015

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
Yes - TritonX-100
Specification
Brain
Blocking step
Donkey Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 Jun 02 2015

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mouse Neuroblastoma (Neuro2A))
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
400000 cells
Treatment
10% FBS and fibroblast growth factor
Specification
Mouse Neuroblastoma (Neuro2A)
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 28°C
Username

Abcam user community

Verified customer

提交于 Apr 27 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 29°C
Sample
Mouse Cell (GL261-mouse glioma)
Specification
GL261-mouse glioma
Permeabilization
Yes - 0.3% TritonX-100 in 1X TBS
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 Nov 28 2014

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Non-reduced Denaturing
Sample
Cow Tissue lysate - whole (Subcutaneous Adipose)
Specification
Subcutaneous Adipose
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

提交于 Nov 08 2013

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (8)
Sample
Rat Cell lysate - whole cell (Primary hippocampal neurons)
Specification
Primary hippocampal neurons
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

提交于 Oct 14 2013

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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