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, 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 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 (ab216772) and 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 : Anti-Dicer antibody [13D6] - ChIP Grade (ab14601) at 1/2000 dilution (incubated overnight at 4°C.)

Lane 1 : NIH 3T3
Lane 2 : A549
Lane 3 : HepG2

Lysates/proteins at 20 µg per lane.

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

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.

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) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (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.

HepG2 cell lysate at 20 µg
Developed using the ECL technique.

Performed under reducing conditions.

Additional bands at: 100 kDa (possible non-specific binding), 60 kDa (possible non-specific binding)

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.

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

Lane 1 : human B cell lymphoma lysate
Lane 2 : human B cell lymphoma lysate infected with control shRNA retrovirus
Lane 3 : human B cell lymphoma lysate infected with Dicer knockdown shRNA virus

Lysates/proteins at 2.5 µg/ml per lane.

Observed band size: 225 kDa why is the actual band size different from the predicted?