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 55 kDa (predicted molecular weight: 51 kDa).
Use at 1-4 µg/mg of lysate.
Use a concentration of 0.5 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.
Use at an assay dependent concentration. PubMed: 20673990
One of the major pre-mRNA-binding proteins. Binds tenaciously to poly(C) sequences. Likely to play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences. Can also bind poly(C) single-stranded DNA.
Contains 3 KH domains.
Arg-296 and Arg-299 are dimethylated, probably to asymmetric dimethylarginine.
Cytoplasm. Nucleus > nucleoplasm. In case of ASFV infection, there is a shift in the localization which becomes predominantly nuclear.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma (left) and mouse squamous cell carcinoma (right) tissues labelling hnRNP K with ab70492 at 1/1000 (0.2µg/ml). Detection: DAB.
IHC image of hnRNP K staining in Human Colon 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 ab70492, 0.5 µg/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.
Immunocytochemistry/ Immunofluorescence - Anti-hnRNP K antibody - ChIP Grade (ab70492)
ICC/IF image of ab70492 stained HeLa 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 (ab70492, 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.
Western blot - hnRNP K antibody (ab70492)
All lanes : Anti-hnRNP K antibody - ChIP Grade (ab70492) 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 Lane 5 : NIH3T3 whole cell lysate at 50 µg
Detection of Human hnRNP K by Immunoprecipitation in Whole cell lysate from HeLa cells (1 mg/IP, 20% of IP loaded) using ab70492 at 3 µg/mg for IP and at 1.0 µg/ml for subsequent WB detection. Lane 2 represents IP IgG control.