Product nameAnti-Cofilin (phospho S3) antibody
See all Cofilin primary antibodies
DescriptionRabbit polyclonal to Cofilin (phospho S3)
Tested applicationsSuitable for: ICC/IF, WB, IHC-FoFr, Flow Cyt, IHC-Pmore details
Species reactivityReacts with: Rat, Dog, Human
Predicted to work with: Mouse
Synthetic peptide derived from a region of human cofilin that contains serine 3. The sequence is conserved in mouse and rat.
- WB: HeLa and A431 cells (untreated and treated with hydrogen peroxide), COS-7, Jurkat, NIH/3T3 and MCF7 cells IHC: Oral carcinoma tissue ICC/IF: Human neuroblastoma cells FC: U-87 MG cells
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage bufferPreservative: 0.05% Sodium azide
Constituents: PBS, 50% Glycerol, 0.1% BSA
PBS (without Mg2+ and Ca2+), BSA (IgG, protease free)
Concentration information loading...
PurityImmunogen affinity purified
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.
|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|
FunctionControls 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 specificityWidely distributed in various tissues.
Sequence similaritiesBelongs to the actin-binding proteins ADF family.
Contains 1 ADF-H domain.
modificationsPhosphorylated on Ser-3 in resting cells.
Cellular localizationNucleus matrix. Cytoplasm > cytoskeleton. Almost completely in nucleus in cells exposed to heat shock or 10% dimethyl sulfoxide.
- Information by UniProt
- 18 kDa phosphoprotein antibody
- CFL 1 antibody
- CFL antibody
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.
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.
All lanes : 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.
This product has been referenced in:
- Zhao F et al. Exendin-4 promotes actin cytoskeleton rearrangement and protects cells from Nogo-A-?20 mediated spreading inhibition and growth cone collapse by down-regulating RhoA expression and activation via the PI3K pathway. Biomed Pharmacother 109:135-143 (2019). Read more (PubMed: 30396070) »
- Yu HX et al. MicroRNA-384 inhibits the progression of esophageal squamous cell carcinoma through blockade of the LIMK1/cofilin signaling pathway by binding to LIMK1. Biomed Pharmacother 109:751-761 (2019). Read more (PubMed: 30551528) »