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Our Abpromise guarantee covers the use of ab31419 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.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|IHC-P||1/50 - 1/100.|
|IP||Use at an assay dependent concentration.|
|WB||1/500 - 1/1000. Predicted molecular weight: 36 kDa.|
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
ChIP analysis using ab31419 binding c-Jun in human endothelial cells (EA.hy926). Cells were cross-linked for 10 minutes with formaldehyde then incubated with undiluted primary antibody for 10 hours at 4°C in 1x ChIP buffer. Protein binding was detected using real-time PCR.
Positive control: Position 89340150-89340297 in chromosome 11 (has a validated c-Jun site).
Negative Control: Igr5 intron 3 (contains no c-Jun binding site).
Paraffin-embedded human breast carcinoma tissue stained for c-Jun with ab31419 at a 1/50 dilution in immunohistochemical analysis.
Left panel: Untreated.
Right panel: Pre-incubated with synthesized peptide.
ICC/IF image of HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling c-Jun (green) with ab31419 at 1 µg/ml. The cells were fixed in 4% PFA (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with ab31419 at 1 µg/ml overnight at +4 °C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) ab150077 used at a 1/1000 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.
c-Jun was immunoprecipitated from HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate using ab31419 at a 1/500 dilution.
Lane 1: Control IgG IP in HEK-293T whole cell lysate.
Lane 2: ab31419 in HEK-293T whole cell lysate.
Lane 3: c-Jun in HEK-293T whole cell lysate 500 µg (Input).
For western blotting an HRP-conjugated swine anti-rabbit polyclonal was used as the secondary antibody.
Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with ab31419 (blue line) at a 1/300 dilution. The cells were prepared as follows: Spin down, wash in FACS buffer 1x, fix, wash 2x, and stain with primary antibody overnight. Buffer was 0.1% sodium azide with FBS in phosphate buffered solution. Cells were fixed using paraformaldehyde and permeabilized using Triton X-100 and NP40. Cells were gated by isolating cell population from plot of SSC-A / FSA-A. The secondary antibody used was a FITC-conjugated Goat anti-Rabbit polyclonal, diluted 1/100.
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