Human Predicted to work with:
Mouse, Rat, Chimpanzee, Macaque monkey, Gorilla, Orangutan
Synthetic peptide corresponding to Human FLASH aa 1950 to the C-terminus conjugated to Keyhole Limpet Haemocyanin (KLH). Database link: Q9UKL3
This antibody gave a positive signal in the following whole cell lysates: HeLa; HEK293; MCF7; MDA MB 361
This antibody gave a positive result when used in the following methanol fixed cell lines: HepG2.
IHC-P: FFPE human breast adenocarcinoma tissue sections.
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.
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 0.1 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 250 kDa (predicted molecular weight: 223 kDa).
Use a concentration of 1 µg/ml.
Participates in TNF-alpha-induced blockade of glucocorticoid receptor (GR) transactivation at the nuclear receptor coactivator level, upstream and independently of NF-kappa-B. Suppresses both NCOA2- and NCOA3-induced enhancement of GR transactivation. Involved in TNF-alpha-induced activation of NF-kappa-B via a TRAF2-dependent pathway. Acts as a downstream mediator for CASP8-induced activation of NF-kappa-B. Required for the activation of CASP8 in FAS-mediated apoptosis. Required for histone gene transcription and progression through S phase.
Phosphorylated upon DNA damage, probably by ATM or ATR.
Cytoplasm. Nucleus. Localizes to discrete nuclear foci.
IHC image of FLASH staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab113899, 0.1µg/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
ICC/IF image of ab113899 stained HepG2 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 ab113899 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
Western blot - Anti-FLASH antibody (ab113899)
All lanes : Anti-FLASH antibody (ab113899) at 1 µg/ml (Milk block 3%)
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 4 : MDA-MB-361 (Human breast adenocarcinoma cell line) Whole Cell Lysate Whole Cell Lysate
Lysates/proteins at 25 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab113899 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.