重组Anti-ATP7A抗体[EPR27493-139] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ATP7A antibody [EPR27493-139] - BSA and Azide free (AB308525)

This data was developed using ab308524, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labelling ATP7A with ab308524 at 1/500 (9.88 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing cytoplasmic staining in SH-SY5Y cell line, no staining was observed in HepG2 cell line. Low expression : HepG2 (PMID : 9693104). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• WB

Lab

Western blot - Anti-ATP7A antibody [EPR27493-139] - BSA and Azide free (AB308525)

This data was developed using the same antibody clone in a different buffer formulation (ab308524).

Western blot : Anti-ATP7A antibody [EPR27493-139] (ab308524) 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, ab308524 was shown to bind specifically to ATP7A. A band was observed at 163 kDa in wild-type A549 cell lysates with no signal observed at this size in ATP7A knockout cell line. To generate this image, wild-type and ATP7A 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-ATP7A antibody [EPR27493-139] (<a href='/products/primary-antibodies/atp7a-antibody-epr27493-139-ab308524'>ab308524</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

ATP7A knockout A549 cell lysate at 20 µg

Lane 3:

HCT116 cell lysate at 20 µg

Lane 4:

A549 Nuclear cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 163 kDa

false

• WB

Supplier Data

Western blot - Anti-ATP7A antibody [EPR27493-139] - BSA and Azide free (AB308525)

This data was produced using ab308524, the same antibody clone but in a different formulation.

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

The molecular weight observed is consistent with the literature (PMID : 16371425).

The identity of the bands between 37 kDa and 100 kDa are unknown.

Samples are non-boiled as boiling may cause protein aggregation.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-ATP7A antibody [EPR27493-139] (<a href='/products/primary-antibodies/atp7a-antibody-epr27493-139-ab308524'>ab308524</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting ATP7A whole cell lysate at 20 µg

Lane 3:

Mouse lung tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 163 kDa

Observed band size: 140-170 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ATP7A antibody [EPR27493-139] - BSA and Azide free (AB308525)

This data was produced using ab308524, the same antibody clone but in a different formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Low expression : HepG2 (PMID : 9693104). The molecular weight observed is consistent with the literature (PMID : 16371425). The lanes 3-5 were developed using a high sensitivity ECL substrate. The identity of the bands between 37 kDa and 100 kDa are unknown. Samples are non-boiled as boiling may cause protein aggregation. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds.

All lanes:

Western blot - Anti-ATP7A antibody [EPR27493-139] (<a href='/products/primary-antibodies/atp7a-antibody-epr27493-139-ab308524'>ab308524</a>) at 1/1000 dilution

Lane 1:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 5:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 163 kDa

Observed band size: 140-170 kDa

false

Exposure time: 180s