The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use at an assay dependent concentration. PubMed: 20053746
Use a concentration of 1 µg/ml.
Use at an assay dependent concentration. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
Transcriptional regulator that binds to the upstream enhancer region (CCAC box) of myoglobin gene. Has a role in myogenic differentiation and in remodeling processes of adult muscles that occur in response to physiological stimuli.
Expressed both developing and adult tissues. In adults, significant expression is seen in tumors of the brain, colon and lymph node.
ab18196 staining in human rectum with colo-rectal cancer, showing staining of the tumour cells (in brown). Paraffin embedded rectal tissue was incubated with ab18196 (1:1000 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab18196 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org
Western blot - FOXK1 antibody - ChIP Grade (ab18196)
All lanes : Anti-FOXK1 antibody (ab18196) at 1/250 dilution
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Nuclear Lysate Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 5 :Jurkat nuclear extract lysate (ab14844) Lane 6 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate Lane 7 :SW480 whole cell lysate (ab3957)
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
ICC/IF image of ab18196 stained Hek293 cells. The cells were 100% methanol fixed (5 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 (ab18196, 1µg/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.
FOXK1 - ChIP Grade was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to FOXK1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18196. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 96kDa:FOXK1.