The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use 1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 1 µg/ml. Detects a band of approximately 64 kDa (predicted molecular weight: 51 kDa).
Use at an assay dependent concentration. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
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.
Formalin-fixed, paraffin embedded tissue sections from human normal colon or human colon carcinoma stained with ab23644 mouse monoclonal to hnRNP K. hnRNP K expression is increased in the tumour tissue.
Immunocytochemistry/ Immunofluorescence - Anti-hnRNP K antibody [F45 P9 C7] (ab23644)
ICC/IF image of ab23644 stained human HepG2 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab23644, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, HEK 293 and MCF7 cells.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP K antibody [F45 P9 C7] (ab23644)This image is courtesy of an Abreview submitted by Satyendra Tripathi
ab23644 staining hnRNP K in Human head and neck tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 1 hour at 25°C; antigen retrieval was by heat mediation with Tris EDTA (pH9). Samples were incubated with primary antibody (1 µg/ml in TBST) for 16 hours at 4°C. An undiluted HRP-conjugated Rabbit anti-mouse polyclonal was used as the secondary antibody.
Flow Cytometry - Anti-hnRNP K antibody [F45 P9 C7] (ab23644)
Overlay histogram showing HeLa cells stained with ab23644 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab23644, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
Immunocytochemistry/ Immunofluorescence - Anti-hnRNP K antibody [F45 P9 C7] (ab23644)Image from Meyerowitz J et al., Mol Neurodegener. 2011 Aug 8;6:57. Fig 10.; doi:10.1186/1750-1326-6-57; 8 August 2011, Molecular Neurodegeneration 2011, 6:57
Immunofluorescence analysis of SH-SY5Y cells staining hnRNP K with ab23644.
Cells were fixed with paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After blocking for 1 hour with 10% normal goat serum, cells were incubated with primary antibody (1/200) overnight at 4°C. An AlexaFluor®-conjugated goat anti-mouse IgG (1/500) was used as the secondary antibody.
Kumar M et al. Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer. J Transl Med13:285 (2015).
Read more (PubMed: 26318153) »
Chauhan SS et al. Prediction of recurrence-free survival using a protein expression-based risk classifier for head and neck cancer. Oncogenesis4:e147 (2015).
Read more (PubMed: 25893634) »