WB: Jurkat and SHSY5Y whole cell lysates.
IHC-P: FFPE human breast adenocarcinoma tissue.
ICC/IF: MCF7 cells.
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
pH: 7.40 Preservative: 0.02% Sodium azide Constituent: PBS Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 20 kDa (predicted molecular weight: 18 kDa).
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.
Widely distributed in various tissues.
Belongs to the actin-binding proteins ADF family. Contains 1 ADF-H domain.
Phosphorylated on Ser-3 in resting cells.
Nucleus matrix. Cytoplasm > cytoskeleton. Almost completely in nucleus in cells exposed to heat shock or 10% dimethyl sulfoxide.
ICC/IF image of ab100836 stained MCF7 cells. The cells were 4% formaldehyde 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 (ab100836, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) HeLa cells at 5µg/ml,.
Western blot - Anti-Cofilin (phospho S3) antibody (ab100836)
All lanes : Anti-Cofilin (phospho S3) antibody (ab100836) at 1 µg/ml
Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 2 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/ml Lane 4 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate with Immunizing peptide at 1 µg/ml Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with Non modified peptide at 1 µg/ml Lane 6 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate with Non modified peptide at 1 µg/ml
Lysates/proteins at 15 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
IHC image of ab100836 staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab100836, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ab100836 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.