Human PINK1 knockout HEK-293T cell line

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Human PINK1 knockout HEK-293T cell line (AB266393)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PINK1 KO HEK-293T (PINK1 knockout human embryonic kidney epithelial cell), ab266393 cells labelling PINK1 with ab323807 at 1/50 (10.34 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 g/ml dilution (Green).

Confocal image showing increased mitochondrial staining in wildtype HEK-293T cells treated with 10 μM Valinomycin for 24 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 g/ml dilution.

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/200 2.5 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Counterstain Secondary antibody only control : Secondary antibody is ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 2 g/ml dilution.

• WB

Lab

Western blot - Human PINK1 knockout HEK-293T cell line (AB266393)

False colour image of Western blot : Anti-PINK1 antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to PINK1. A band was observed at 63 kDa in wild-type 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 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Anti-PINK1 antibody at 1/1000 dilution

Lane 1:

Wild-type HEK-293T Vehicle Control CCCP (0 μM, 24 h) cell lysate at 20 µg

Lane 2:

Wild-type HEK-293T Treated CCCP (10 μM, 24 h) cell lysate at 20 µg

Lane 3:

PINK1 knockout HEK-293T Vehicle Control CCCP (0 μM, 24 h) cell lysate at 20 µg

Lane 4:

PINK1 knockout HEK-293T Treated CCCP (10 μM, 24 h) cell lysate at 20 µg

Lane 5:

HeLa cell lysate at 20 µg

Lane 6:

Daudi cell lysate at 20 µg

Secondary

Lanes 1 - 6:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 6:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 63 kDa

false

• Sanger seq

Lab

Sanger Sequencing - Human PINK1 knockout HEK-293T cell line (AB266393)

Sequencing chromatogram displaying sequence edit in exon 1

• Sanger seq

Unknown

Sanger Sequencing - Human PINK1 knockout HEK-293T cell line (AB266393)

Homozygous : 10 bp deletion in exon1

• Cell Culture

Lab

Cell Culture - Human PINK1 knockout HEK-293T cell line (AB266393)

Representative images PINK1 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS M5000 microscope.