Anti-Dicer antibody [13D6] - ChIP Grade

6 product images

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  Flow Cytometry - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  Overlay histogram showing HEK293 cells stained with ab14601 (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 (ab14601, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (Mouse IgG2a, Kappa Monoclonal [B12/8] - Isotype Control ab91361, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

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  This image is courtesy of Elliot Epner MD PhD, Oregon Health and Science University, Portland

  ChIP - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  ChIP analysis of Dicer protein binding at chicken ß-globin regulatory elements. Chromatin from human erythroleukemia (K562) cells containing chicken chromosomes with normal (FRK2) or mutant (HS1-K) ß-globin loci and chicken DT40 cells was immunprecipitated with antibodies to Dicer (ab14601). PCR analysis of immunoprecipitated chromatin was carried out using primers to the chicken ß-globin regulatory elements. Mouse IgG served as control. 10% of input DNA was utilized.

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  Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: Dicer knockout HAP1 cell lysate (20 μg)
  

  Lanes 1 and 2: Merged signal (red and green). Green - ab14601 observed at 240 kDa. Red - loading control, Anti-GAPDH antibody [EPR16891] - Loading Control ab181602, observed at 37 kDa.

  
  ab14601 was shown to specifically react with Dicer in wild-type HAP1 cells. No band was observed when Dicer knockout samples were used. Wild-type and Dicer knockout samples were subjected to SDS-PAGE. ab14601 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

  All lanes: Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  Predicted band size: 218 kDa

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  Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  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 before being incubated with ab14601 overnight at 4°C. Antibody binding was detected using IR-labelled goat anti-mouse Ab at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  All lanes: Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601) at 1/2000 dilution

  Lane 1: NIH 3T3 at 20 µg

  Lane 2: A549 at 20 µg

  Lane 3: HepG2 at 20 µg

  Secondary

  All lanes: Goat anti-Mouse Green at 1/10000 dilution

  Predicted band size: 218 kDa

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  Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  All lanes: HepG2 cell lysate at 20 µg

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 218 kDa

  Observed band size: 100 kDa, 60 kDa

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  This image is courtesy of Jing Huang and Elliot Epner Oregon Health and Science University

  Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

  All lanes: Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (ab14601) at 2.5 µg/mL

  Lane 1: human B cell lymphoma lysate at 2.5 µg/mL

  Lane 2: human B cell lymphoma lysate infected with control shRNA retrovirus at 2.5 µg/mL

  Lane 3: human B cell lymphoma lysate infected with Dicer knockdown shRNA virus at 2.5 µg/mL

  Predicted band size: 218 kDa

  Observed band size: 225 kDa