Anti-PINK1 antibody [MJF-R32-7] - BSA and Azide free

• WB

Lab

Western blot - Anti-PINK1 antibody [MJF-R32-7] - BSA and Azide free (AB300624)

This data was developed using ab300623, the same antibody clone in a different buffer formulation.

Western blot : Anti-PINK1 antibody [MJF-R32-7] (ab300623) staining at 1/1000 dilution, shown in black; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta.

In Western blot, ab300623 was shown to bind specifically to PINK1. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% 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 before development with a high-sensitivity ECL substrate kit and imaged with 20 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

In Western blot, ab300623 was shown to bind specifically to PINK1. A band was observed at 63kDa in wild-type SH-SY5Y treated with CCCP cell lysates with no signal observed at this size in PINK1 knockout cell line (ab280876).

All lanes:

Western blot - Anti-PINK1 antibody [MJF-R32-7] (<a href='/en-us/products/primary-antibodies/pink1-antibody-mjf-r32-7-ab300623'>ab300623</a>)

Lane 1:

Wild-type SH-SY5Y treated CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (10 &mu;M, 24 h) cell lysate at 20 µg

Lane 2:

Wild-type SH-SY5Y control CCCP (0 &mu;M, 24 h) cell lysate at 20 µg

Lane 3:

PINK1 knockout SH-SY5Y (<a href='/en-us/products/cell-lines/human-pink1-knockout-sh-sy5y-cell-line-ab280876'>ab280876</a>) treated CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (10 uM, 24 h) cell lysate at 20 µg

Lane 4:

Western blot - Human PINK1 knockout SH-SY5Y cell line (<a href='/en-us/products/cell-lines/human-pink1-knockout-sh-sy5y-cell-line-ab280876'>ab280876</a>) at 20 µg

Lane 5:

Wild-type HEK-293 Treated CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (10 &mu;M, 24 h) cell lysate at 20 µg

Lane 6:

PINK1 knockout HEK-293 Treated CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (10 &mu;M, 24 h) cell lysate at 20 µg

Secondary

All lanes:

HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 63 kDa

Observed band size: 63 kDa

true

• WB

Supplier Data

Western blot - Anti-PINK1 antibody [MJF-R32-7] - BSA and Azide free (AB300624)

This data was developed using ab300623, the same antibody clone in a different buffer formulation.  Anti-PINK1 antibody [MJF-R32-7] (ab300623) staining at 1/1000 dilution, shown in black; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. ab300623 was shown to bind specifically to PINK1. A band was observed at 60 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in PINK1 knockout cell line ab266393 (knockout cell lysate ab257030). To generate this image, wild-type and PINK1 knockout HEK-293 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 before development with Optiblot (ECL reagent ab133456) and imaged with 4 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PINK1 antibody [MJF-R32-7] (<a href='/en-us/products/primary-antibodies/pink1-antibody-mjf-r32-7-ab300623'>ab300623</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 Vehicle Control CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (0 µM, 24 h) cell lysate at 20 µg

Lane 2:

Wild-type HEK-293 Treated CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (10 µM, 24 h) cell lysate at 20 µg

Lane 3:

PINK1 knockout HEK-293 Vehicle Control CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (0 µM, 24 h) cell lysate at 20 µg

Lanes 3 - 4:

Western blot - Human PINK1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-pink1-knockout-hek-293t-cell-line-ab266393'>ab266393</a>)

Lane 4:

PINK1 knockout HEK-293 Treated CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a> (10 µM, 24 h) cell lysate at 20 µg

Secondary

All lanes:

HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 63 kDa

false

• WB

Supplier Data

Western blot - Anti-PINK1 antibody [MJF-R32-7] - BSA and Azide free (AB300624)

All lanes:

Western blot - Anti-PINK1 antibody [MJF-R32-7] (<a href='/en-us/products/primary-antibodies/pink1-antibody-mjf-r32-7-ab300623'>ab300623</a>) at 1/1000 dilution

All lanes:

Rat testis tissue 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

false