Anti-ITCH/AIP4 antibody [32/Itch] - BSA and Azide free

• WB

Lab

Western blot - Anti-ITCH/AIP4 antibody [32/Itch] - BSA and Azide free (AB280218)

This data was developed using ab280211, the same antibody clone in a different buffer formulation.

ab280211 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 ab280211 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 [32/Itch] (<a href='/en-us/products/primary-antibodies/itch-aip4-antibody-32-itch-ab280211'>ab280211</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 100 µg

Lane 2:

ITCH knock-out HeLa  lysate at 100 µg

false

• WB

Supplier Data

Western blot - Anti-ITCH/AIP4 antibody [32/Itch] - BSA and Azide free (AB280218)

This data was developed using ab280211, the same antibody clone in a different buffer formulation.

Blocking and Diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1 - 4 : Merged signal (red and green). Green - ab280211 observed at 120 kDa. Red - loading control ab181602 (Rabbit monoclonal [EPR16891] to GAPDH) observed at 36 kDa.

Lanes 1-2 : ab280211 Anti-ITCH antibody was shown to react with ITCH in HeLa cells in Western blot. Loss of signal was observed when ITCH knockout sample was used. Wild-type and ITCH knockout samples were subjected to SDS-PAGE.

ab280211 and Anti-GAPDH antibody [EPR16891] (ab181602) were incubated at 4℃ overnight at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680CW) (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800RD) (ab216772) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

The band around 75kDa is the degradation form. 50kDa band is the non-specific band according to KO validation.

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [32/Itch] (<a href='/en-us/products/primary-antibodies/itch-aip4-antibody-32-itch-ab280211'>ab280211</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

ITCH knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Lane 4:

Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Mouse IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 103 kDa

Observed band size: 120 kDa

false

• WB

Supplier Data

Western blot - Anti-ITCH/AIP4 antibody [32/Itch] - BSA and Azide free (AB280218)

This data was developed using ab280211, the same antibody clone in a different buffer formulation.

Blocking and Diluting buffer and concentration : 5% NFDM/TBST

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 11782481).

The band around 75kDa is the degradation form. 50kDa band is the non-specific band according to KO validation.

Exposure time Lane 1-2/4-5 : 3 minutes Lane 3/6 : 1 minute

All lanes:

Western blot - Anti-ITCH/AIP4 antibody [32/Itch] (<a href='/en-us/products/primary-antibodies/itch-aip4-antibody-32-itch-ab280211'>ab280211</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 10 µg

Lane 2:

Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 10 µg

Lane 3:

Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 10 µg

Lane 4:

C6 (rat glial tumor glial cell), whole cell lysate at 10 µg

Lane 5:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 103 kDa

Observed band size: 120 kDa

false