The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/2000 - 1/10000. Detects a band of approximately 139, 160 kDa (predicted molecular weight: 139 kDa).
Use at 2-5 µg/mg of lysate.
1/200. Use with paraformaldehyde-fixed cells permeabilized with 0.5 % Triton X-100.
1/500 - 1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Required for normal progression of S-phase. Involved in the circadian rhythm autoregulatory loop. Negatively regulates CLOCK-NPAS2/BMAL1-induced transactivation of PER1 possibly via translocation of PER1 into the nucleus. Promotes TIPIN nuclear localiZation. Involved in cell survival after DNA damage or replication stress. May be specifically required for the ATR-CHK1 pathway in the replication checkpoint induced by hydroxyurea or ultraviolet light. May also play an important role in epithelial cell morphogenesis and formation of branching tubules.
Expressed in all tissues examined including brain, heart, lung, liver, skeletal muscle, kidney, placenta, pancreas, spleen, thymus and testis. Highest levels of expression in placenta, pancreas, thymus and testis.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Timeless with ab72458 at 1/400 (0.5µg/ml). A DyLight® 594-conjugated goat anti-rabbit IgG (H+L) (1/100) was used as the secondary antibody.
Western blot - Timeless antibody (ab72458)
All lanes : Anti-Timeless antibody (ab72458) at 0.04 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg
Predicted band size: 139 kDa Observed band size: 139 kDa Additional bands at: 160 kDa. We are unsure as to the identity of these extra bands.
Immunoprecipitation - Timeless antibody (ab72458)
HeLa whole cell lysate, (1 mg for IP, 20% of IP loaded) with ab 72458 used for IP at 3 µg/mg lysate, and for subsequent WB at 0.1 µg/ml.
Detection: Chemiluminescence with an exposure time of 3 seconds.
Immunocytochemistry/ Immunofluorescence - Timeless antibody (ab72458)Image courtesy of an abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
ab72458 (1/200) staining Timeless in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 and counterstained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.
IHC image of ab72458 staining Timeless in Human Tonsil 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 ab72458, 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.
Calì F et al. Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway. Nucleic Acids Res44:705-17 (2016).
Read more (PubMed: 26503245) »
Mazzoccoli G et al. Analysis of clock gene-miRNA correlation networks reveals candidate drivers in colorectal cancer. Oncotarget7:45444-45461 (2016).
Read more (PubMed: 27323779) »