Anti-CEP290 antibody

• ICC

Lab

Immunocytochemistry - Anti-CEP290 antibody (AB85728)

ICC/IF image of ab85728 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab85728, 5μg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• ICC

Supplier Data

Immunocytochemistry - Anti-CEP290 antibody (AB85728)

Immunocytochemistry/Immunofluorescence analysis of NBF-fixed asynchronous HeLa cells labelling CEP290 with ab85728 at 1/500. A DyLight® 594-conjugated goat anti-rabbit IgG (1/100) was used as the secondary antibody.

• IP

Unknown

Immunoprecipitation - Anti-CEP290 antibody (AB85728)

Detection of Human CEP290 in Immunoprecipitates of Whole cell lysate from HeLa cells (1 mg for IP, 20% of IP loaded) using ab85728 at 3 µg/mg lysate for IP (Lane 1) and at 1 µg/ml for subsequent Western blot detection. Lane 2 represents control IgG IP. Detection : Chemiluminescence with an exposure time of 10 seconds.

All lanes:

Immunoprecipitation - Anti-CEP290 antibody (ab85728)

Predicted band size: 290 kDa

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• WB

Unknown

Western blot - Anti-CEP290 antibody (AB85728)

All lanes:

Western blot - Anti-CEP290 antibody (ab85728) at 0.1 µg/mL

Lane 1:

Whole cell lysate from HeLa cells at 50 µg

Lane 2:

Whole cell lysate from HeLa cells at 15 µg

Lane 3:

Whole cell lysate from HeLa cells at 5 µg

Lane 4:

Whole cell lysate from 293T cells at 50 µg

Predicted band size: 290 kDa

Observed band size: 100 kDa,200 kDa,270 kDa

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Exposure time: 30s

• WB

Lab

Western blot - Anti-CEP290 antibody (AB85728)

Western blot : Anti-CEP290 antibody (ab85728) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab85728 was shown to bind specifically to CEP290. A band was observed at 290 kDa in wild-type A549 cell lysates with no signal observed at this size in CEP290 knockout cell line. To generate this image, wild-type and CEP290 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CEP290 antibody (ab85728) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 40 µg

Lane 2:

CEP290 knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type A549 ab288558 cell lysate at 10 µg

Lane 4:

CEP290 knockout A549 STN-436528 cell lysate at 40 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 290 kDa

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