All lanes : Anti-hnRNP K antibody (ab18195) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line)
Lane 3 : Testis (Mouse) Tissue Lysate
Lane 4 : Ovary (Mouse) Tissue Lysate
Lane 5 : PC12 (Rat adrenal pheochromocytoma cell line)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 51 kDa
Observed band size: 65 kDa

why is the actual band size different from the predicted?

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 ab18195, 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.

ICC/IF image of ab18195 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18195, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Panel A (left) shows localisation of the antibody (green) to specific foci in the nucleus, panel B (right) does not show the Alexa Fluor® 488 channel for comparison.

ab18195 staining hnRNP K in HeLa cells treated with PD 98059 (ab120234), by ICC/IF. Changes in localization of hnRNP K (translocation from cytoplasm to nucleous) correlates with increased concentration of hnRNP K, as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab120234 ( hnRNP K) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab18195(5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.