Synthetic peptide conjugated to KLH derived from within residues 1500 to the C-terminus of Human Jarid1C/ SMCX.
(Peptide available as ab35501.)
Our Abpromise guarantee covers the use of ab34718 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use at an assay dependent concentration.|
|IHC-FoFr||Use at an assay dependent concentration.|
|WB||1/250. Detects a band of approximately 180 kDa (predicted molecular weight: 176 kDa).
Abcam recommends using milk as the blocking agent.
|ICC/IF||Use a concentration of 1 µg/ml.|
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: KDM5C knockout HAP1 whole cell lysate (20 µg)
Lane 3: Hek 293 whole cell lysate (20 µg)
Lane 4: U2OS whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab34718 observed at 175 kDa. Red - loading control, ab18058, observed at 120 kDa.
ab34718 was shown to recognize KDM5C in wild type cells as signal was lost at the expected MW in KDM5C knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and KDM5C knockout samples were subjected to SDS-PAGE. Ab34718 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 250 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
PFA-fixed, 0.5% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for KDM5C / Jarid1C / SMCX (green) using ab34718 at 1/200 dilution in ICC/IF. Counter-stained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab34718 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
ICC/IF image of ab34718 stained MCF-7 cells. The cells were 4% formaldehyde 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 ab34718 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.
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