The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 130 kDa (predicted molecular weight: 119 kDa).
Use a concentration of 5 µg/ml.
Functions in nuclear protein import as nuclear transport receptor. Serves as receptor for nuclear localization signals (NLS) in cargo substrates. Is thought to mediate docking of the importin/substrate complex to the nuclear pore complex (NPC) through binding to nucleoporin and the complex is subsequently translocated through the pore by an energy requiring, Ran-dependent mechanism. At the nucleoplasmic side of the NPC, Ran binds to the importin, the importin/substrate complex dissociates and importin is re-exported from the nucleus to the cytoplasm where GTP hydrolysis releases Ran. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus (By similarity). Mediates the nuclear import of RPS3A. In vitro, mediates the nuclear import of human cytomegalovirus UL84 by recognizing a non-classical NLS.
Belongs to the importin beta family. Contains 6 HEAT repeats. Contains 1 importin N-terminal domain.
Importin4 antibody ab28387 detected a 130 kDa doublet in Hela B lysate that is likely to correspond to Importin4.
Staining of tubulin in the same lanes is shown as a loading control. The antibody was also able to detect recombinant Importin4 protein but not recombinant Importin5 protein (not shown).
Western blot - Anti-Importin4 antibody (ab28387)
All lanes : Anti-Importin4 antibody (ab28387) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 :HeLa nuclear extract lysate (TPA stimulated 2 hr) (ab14653)
Lysates/proteins at 20 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size: 119 kDa Observed band size: 119 kDa Additional bands at: 116 kDa. We are unsure as to the identity of these extra bands.
ICC/IF image of ab28387 stained HeLa 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 (ab28387, 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.
IHC image of ab28387 staining in human skin 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 ab28387, 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.