Anti-Dicer 抗体 [13D6] - ChIP Grade

• WB

Lab

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, 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:

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

Predicted band size: 218 kDa

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• Flow Cyt

Unknown

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) (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.

• ChIP

Collaborator

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.

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

• WB

Lab

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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• WB

Supplier Data

Western blot - Anti-Dicer antibody [13D6] - ChIP Grade (AB14601)

All lanes:

HepG2 cell lysate at 20 µg

Predicted band size: 218 kDa

Observed band size: 100 kDa,60 kDa

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• WB

Supplier Data

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

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