Anti-Cofilin (phospho S3)抗体
参阅全部 Cofilin-1 一抗
兔多克隆抗体to Cofilin (phospho S3)
Rabbit
适用于: WB, ICC/IF, Flow Cytmore details
与反应: Mouse, Rat, Dog, Human, African green monkey
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Liquid
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
pH: 7.3
Preservative: 0.05% Sodium azide
Constituents: PBS, 50% Glycerol, 0.1% BSA
PBS (without Mg2+ and Ca2+), BSA (IgG, protease free)
浓度
50 µl 浓度为 0.5 mg/ml
Immunogen affinity purified
多克隆
IgG
Abpromise™承诺保证使用ab12866于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。
| 应用 | Ab评论 | 说明 |
|---|---|---|
| WB | (6) | 1/1000. Detects a band of approximately 20 kDa. |
| ICC/IF | (3) | 1/250. |
| Flow Cyt | Use 3-5µg for 106 cells. |
Entrez Gene: 1072 Human
Entrez Gene: 12631 Mouse
Omim: 601442 Human
SwissProt: P23528 Human
SwissProt: P18760 Mouse
Unigene: 170622 Human
Unigene: 329655 Mouse
Unigene: 11675 Rat
18 kDa phosphoprotein antibody
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Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (ab12866)
Immunofluorescence analysis of Phospho-Cofilin pSer3 was done on 70% confluent log phase PC-3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab12866 at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Rhodamine Phalloidin (1:300). Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
Western blot - Anti-Cofilin (phospho S3) antibody (ab12866)
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
Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (ab12866)
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.
Immunocytochemistry/ Immunofluorescence - Anti-Cofilin (phospho S3) antibody (ab12866)This image is courtesy of an anonymous abreview.
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.
Western blot - Anti-Cofilin (phospho S3) antibody (ab12866)
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 (Mouse embryo fibroblast cell line) 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
All lanes : Goat anti-rabbit IgG (H+L), HRP conjugate at 1/2500 dilution
Observed band size: 18 kDawhy is the actual band size different from the predicted?
Flow Cytometry - Anti-Cofilin (phospho S3) antibody (ab12866)
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.
