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Synthetic peptide conjugated to KLH derived from within residues 600 to the C-terminus of Human DDX5.
(Peptide available as ab22412.)
Our Abpromise guarantee covers the use of ab21696 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||1/1000 - 1/5000. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
Perform heat mediated antigen retrieval via the pressure cooker method with citrate buffer pH 6.0 before commencing with IHC staining protocol.
|ICC/IF||Use a concentration of 1 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 69 kDa (predicted molecular weight: 69 kDa).|
Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: DDX5 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab21696 observed at 69 kDa. Red - loading control, ab9484, observed at 37 kDa.
ab21696 was shown to specifically react with DDX5 in wild-type HAP1 cells as signal was lost in DDX5 knockout cells. Wild-type and DDX5 knockout samples were subjected to SDS-PAGE. Ab21696 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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.
ab21696 recognizes a clear, strong band at ~ 69kDa corresponding to DDX5. A number of other, non specific bands are weakly recognized by the antibody. All bands are quenched by the addition of the blocking peptide.
ICC/IF image of ab21696 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 10 min) and incubated with the antibody (ab21696, 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-iTTM 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).
ab21696 localises to the nucleus as expected. We believe the small amount of signal seen skirting the nucleus is due to inefficient fixation of the sample.
ab21696 staining DDXA in paraffin embedded Human adrenal gland. The tissue was incubated with ab21696 (1/3000 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
Further results for this antibody can be found at www.proteinatlas.org
ab21696 staining mouse adrenal tissue sections by IHC-P. Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking. The primary antibody was diluted 1/1000 and incubated with the sample for 40 minutes at 20°C. A HRP conjugated goat anti-rabbit antibody was used as the secondary.
ab21696 staining human anal cancer tissue sections by IHC-P. Sections were formaldehyde fixed and subjected to heat mediated antigen retreival in citrate buffer (pH 6) prior to blocking in a commercially available blocking agent for 20 minutes at 20°C. The primary anibody was diluted 1/1000 and incubated with the sample for 40 minutes at 20°C. A HRP-conjugated goat anti-rabbit antibody was used as the secondary.
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