Anti-Cofilin (phospho S3) antibody (ab12866)

Overview

  • Product name
    Anti-Cofilin (phospho S3) antibody
    See all Cofilin primary antibodies
  • Description
    Rabbit polyclonal to Cofilin (phospho S3)
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-FoFr, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Rat, Dog, Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic peptide derived from a region of human cofilin that contains serine 3. The sequence is conserved in mouse and rat.

  • Positive control
    • MDCK cells treated with staurosporine; Jurkat cells treated with hydrogen peroxide.

Properties

Applications

Our Abpromise guarantee covers the use of ab12866 in the following tested applications.

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

Application Abreviews Notes
ICC/IF Use a concentration of 2 - 3 µg/ml.
WB Use at an assay dependent concentration. Detects a band of approximately 20 kDa.
IHC-FoFr Use at an assay dependent concentration. PubMed: 17652593
Flow Cyt Use 3-5µg for 106 cells.
IHC-P Use at an assay dependent concentration. PubMed: 20797537

Target

  • Function
    Controls reversibly actin polymerization and depolymerization in a pH-sensitive manner. It has the ability to bind G- and F-actin in a 1:1 ratio of cofilin to actin. It is the major component of intranuclear and cytoplasmic actin rods.
  • Tissue specificity
    Widely distributed in various tissues.
  • Sequence similarities
    Belongs to the actin-binding proteins ADF family.
    Contains 1 ADF-H domain.
  • Post-translational
    modifications
    Phosphorylated on Ser-3 in resting cells.
  • Cellular localization
    Nucleus matrix. Cytoplasm > cytoskeleton. Almost completely in nucleus in cells exposed to heat shock or 10% dimethyl sulfoxide.
  • Information by UniProt
  • Database links
  • Alternative names
    • 18 kDa phosphoprotein antibody
    • CFL 1 antibody
    • CFL antibody
    • CFL1 antibody
    • COF1_HUMAN antibody
    • Cofilin 1 antibody
    • Cofilin 1 non muscle antibody
    • Cofilin antibody
    • Cofilin non muscle isoform antibody
    • Cofilin-1 antibody
    • epididymis secretory protein Li 15 antibody
    • HEL-S-15 antibody
    • non-muscle isoform antibody
    • p18 antibody
    see all

Anti-Cofilin (phospho S3) antibody images

  • Peptide Competition and Phosphatase Treatment
    Lysates prepared from MDCK cells treated with staurosporine (1) or left untreated (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-5) or treated with lambda phosphatase (6), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with the ab12866 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4) or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that only the peptide corresponding to cofilin [pS3] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the anti
  • ICC/IF image of ab12866 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab12866, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of oral squamous cell carcinomas (OSCCs) surgical margin labeling Cofilin (phospho S3) with ab12866 at 1/300 dilution. After deparaffinization in xylene and rehydration in graded ethanol, antigen epitope retrieval was performed using 10 mM citrate buffer, pH 6.0 in a vapor cooker. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. Rabbit polyclonal anti-cofilin (phospho S3) (ab12866, 1/300) was incubated overnight at 8°C followed by addition of the secondary antibody and streptavidin-biotin peroxidase. Color of reaction product was developed by 3,3′-diaminobenzidine (DAB) and counterstaining was performed with hematoxylin.

    Nuclear staining for Cofilin (phospho S3) is evident in the more basal layers of epithelium in surgical margin tissue compared to OSCCs tissue.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of oral squamous cell carcinomas (OSCCs) labeling Cofilin (phospho S3) with ab12866 at 1/300 dilution. After deparaffinization in xylene and rehydration in graded ethanol, antigen epitope retrieval was performed using 10 mM citrate buffer, pH 6.0 in a vapor cooker. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. Rabbit polyclonal anti-cofilin (phospho S3) (ab12866, 1/300) was incubated overnight at 8°C followed by addition of the secondary antibody and streptavidin-biotin peroxidase. Color of reaction product was developed by 3,3′-diaminobenzidine (DAB) and counterstaining was performed with hematoxylin.

  • Immunocytochemistry/ Immunofluorescence analysis of human neuroblastoma cells labeling Cofilin (phospho S3) with ab12866 at 1/500 dilution. Cells were fixed in paraformaldehyde, permeabilized for 15 minutes in 0.01% Triton X-100, blocked using 5% serum for 30 minutes at 20°C, then incubated with ab12866 at a 1/500 dilution for 2 hours at 20°C. The secondary used was a Dylight 488 conjugated donkey anti-rabbit IgG (H+L) used at a 1/500 dilution.

    See Abreview

  • All lanes : Anti-Cofilin (phospho S3) antibody (ab12866) at 1 µg/ml

    Lane 1 : HeLa whole cell extract
    Lane 2 : HeLa treated for overnight with 150 uM of H2O2 whole cell extract
    Lane 3 : HeLa treated for overnight with 3 uM of Staurosporine whole cell extract
    Lane 4 : COS-7 whole cell extract
    Lane 5 : NIH/3T3 whole cell extract
    Lane 6 : MCF7 whole cell extract
    Lane 7 : Rat Skeletal Muscle whole cell extract
    Lane 8 : A-431 whole cell extract
    Lane 9 : A-431 treated for overnight with 150 uM of H2O2 whole cell extract
    Lane 10 : Jurkat whole cell extract
    Lane 11 : Jurkat treated for overnight with 3 uM of Staurosporine whole cell extract

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat anti-rabbit IgG (H+L), HRP conjugate at 1/2500 dilution

    Observed band size : 18 kDa (why is the actual band size different from the predicted?)
  • Flow Cytometry analysis of U-87 MG cells labeling Cofilin (phospho S3) with ab12866. 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 Anti-Cofilin (phospho S3) antibody (ab12866, red) or with rabbit isotype control (pink) 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 at a dilution of 1/400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

     

References for Anti-Cofilin (phospho S3) antibody (ab12866)

This product has been referenced in:
  • Lee HJ  et al. Fluid shear stress activates YAP1 to promote cancer cell motility. Nat Commun 8:14122 (2017). WB ; Human . Read more (PubMed: 28098159) »
  • Jitsuki S  et al. Nogo Receptor Signaling Restricts Adult Neural Plasticity by Limiting Synaptic AMPA Receptor Delivery. Cereb Cortex 26:427-39 (2016). WB ; Mouse . Read more (PubMed: 26472557) »

See all 32 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Primary cortical neuronal cells)
Permeabilization
Yes - 0.2% Triton X-100
Specification
Primary cortical neuronal cells
Blocking step
casein solution + Tween 20 as blocking agent for 20 minute(s) · Concentration: 0.2% · Temperature: 25°C
Fixative
Ethanol
Username

Abcam user community

Verified customer

Submitted Aug 18 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Nervous Tissue (Nucleus Accumbens))
Gel Running Conditions
Reduced Denaturing (10% gel)
Loading amount
40 µg
Specification
Nervous Tissue (Nucleus Accumbens)
Blocking step
Li-Cor Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 21°C
Username

Mr. Jeffrey Lenz

Verified customer

Submitted Jan 27 2016

Application
Western blot
Loading amount
75 µg
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Mouse Tissue lysate - whole (brain tissue)
Specification
brain tissue
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Nov 10 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Tissue lysate - whole (Cerebellum, Brain)
Specification
Cerebellum, Brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

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Verified customer

Submitted Apr 02 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing
Sample
Rat Tissue lysate - whole (Brain)
Specification
Brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Jun 24 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Human Cell lysate - whole cell (Neuroblastoma)
Specification
Neuroblastoma
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Jun 24 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Neuroblastoma)
Specification
Neuroblastoma
Fixative
Formaldehyde
Permeabilization
Yes - 0.1% Trition X
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 21°C
Username

Abcam user community

Verified customer

Submitted May 13 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Neuroblastoma)
Specification
Neuroblastoma
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.01% Triton X
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Sep 04 2012

Thank you very much for your call today and for your questions about ab12866.

I've further discussed the antibody and proposed staining method with my colleague, and even though we have not tested the antibody in exactly this way,your protoco...

Read More

Thank you for your inquiry.
That antibody is our ab12866, rabbit polyclonal to Cofilin (phospho S3).
http://www.abcam.com/Cofilin-phospho-S3-antibody-ab12866.html
We have that reference listed on our publications page.
http://www.ab...

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