Anti-PINK1 antibody [EPR29146-340]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PINK1 antibody [EPR29146-340] (AB323807)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PINK1 antibody [EPR29146-340] (AB323807)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SK-OV-3(human ovarian cancer epithelial cell) 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 mitochondrial staining in SK-OV-3 cells (shown in green) treatment with 30 μM CCCP for 6 hours. 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

Supplier Data

Western blot - Anti-PINK1 antibody [EPR29146-340] (AB323807)

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

Performed under reducing conditions.

In Western blot, ab323807 was shown to bind specifically to PINK1. Target of interest was observed at 62 kDa in wild-type HEK-293 cell lysates (lane 2) with no signal observed at this size in PINK1 knockout cell line (lane 4) (lane 4, knockout cell line ab266393).

The identity of the higher MW band at approximately 250 kDa (in lanes 1-4) is unknown.

The expression of PINK1 is upregulated in response to CCCP treatment (PMID : 22724072).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-PINK1 antibody [EPR29146-340] (ab323807) at 1/1000 dilution

Lane 1:

Untreated wild-type HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 80 µg

Lane 2:

Wild-type HEK-293 treated with 30μM CCCP for 6h, whole cell lysate at 80 µg

Lane 3:

Untreated PINK1 knockout HEK-293 whole cell lysate at 80 µg

Lane 4:

PINK1 knockout HEK-293 treated with 30μM CCCP for 6h, whole cell lysate at 80 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

Observed band size: 62 kDa,36 kDa

false

Exposure time: 125s

• WB

Supplier Data

Western blot - Anti-PINK1 antibody [EPR29146-340] (AB323807)

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

Performed under reducing conditions.

In Western blot, ab323807 was shown to bind specifically to PINK1. Target of interest was observed at 62 kDa in wild-type HEK-293 cell lysates (lane 2) with no signal observed at this size in PINK1 knockout cell line (lane 4) (lane 4, knockout cell line ab266393).

The identity of the higher MW band at approximately 250 kDa (in lanes 1-4) is unknown.

The expression of PINK1 is upregulated in response to valinomycin treatment (PMID : 22724072).

The expression of PINK1 is upregulated in response to CCCP treatment (PMID : 22724072).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-4 : 48 seconds, Lanes 5-6 : 10 seconds

All lanes:

Western blot - Anti-PINK1 antibody [EPR29146-340] (ab323807) at 1/1000 dilution

Lane 1:

Untreated wild-type HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HEK-293 treated with 10μM valinomycin for 24h, whole cell lysate at 20 µg

Lane 3:

Untreated PINK1 knockout HEK-293 whole cell lysate at 20 µg

Lane 4:

PINK1 knockout HEK-293 treated with 10μM valinomycin for 24h, whole cell lysate at 20 µg

Lane 5:

Untreated SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate at 20 µg

Lane 6:

SK-OV-3 treated with 30μM CCCP for 6h, whole cell 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

Observed band size: 62 kDa,36 kDa

false

• I-ELISA

Supplier Data

Indirect ELISA - Anti-PINK1 antibody [EPR29146-340] (AB323807)

Indirect ELISA analysis of ab323807 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Human PINK1.

Antigen concentration : 1000 ng/ml

"