Anti-ITCH/AIP4 antibody [EPR4936]

• WB

Lab

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (AB108515)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : ITCH/AIP4 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : K562 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab108515 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab108515 was shown to specifically react with ITCH/AIP4 when ITCH/AIP4 knockout samples were used. Wild-type and ITCH/AIP4 knockout samples were subjected to SDS-PAGE. ab108515 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515)

Predicted band size: 103 kDa

Observed band size: 110 kDa

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• WB

Unknown

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (AB108515)

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

Lane 1:

K562 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

293T cell lysate at 10 µg

Predicted band size: 103 kDa

false

• WB

Lab

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (AB108515)

Lanes 1-4 : Merged signal (red and green). Green - ab108515 observed at 103 kDa. Red - loading control ab8245 observed at 36 kDa.

ab108515 Anti-ITCH/AIP4 antibody [EPR4936] was shown to specifically react with ITCH/AIP4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265338 (knockout cell lysate ab258014) was used. Wild-type and ITCH/AIP4 knockout samples were subjected to SDS-PAGE. ab108515 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ITCH knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ITCH (AIP4) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-itch-aip4-knockout-hela-cell-line-ab265338'>ab265338</a>)

Lane 3:

HAP1 cell lysate at 20 µg

Lane 4:

K-562 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 103 kDa,43 kDa

Observed band size: 103 kDa,57 kDa

false

• WB

Lab

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (AB108515)

ab108515 was shown to react with ITCH in wild-type   cells in Western blot with loss of signal observed in a ITCH knockout cell line. Wild-type   and ITCH knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab108515 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 100 µg

Lane 2:

ITCH knock-out HeLa  lysate at 100 µg

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• WB

Lab

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (AB108515)

Blocking buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [EPR4936] (ab108515) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 103 kDa

Observed band size: 103 kDa

false

Exposure time: 1min