Anti-PINK1 antibody [EPR20730]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PINK1 antibody [EPR20730] (AB216144)

Immunofluorescent analysis of 4 % paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma)(+/- treatment with 10μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, ab141229) for 24 hours) cells labeling PINK1 with ab216144 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells treated with 10μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, ab141229) for 24 hours. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

The negative controls are as follows :
-ve control : PBS, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-PINK1 antibody [EPR20730] (AB216144)

PINK1 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP. ab141229) for 24 hours) whole cell lysate with ab216144 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216144 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution.

Lane 1 : HeLa (CCCP-treated, ab141229) lysate 10 μg (Input).
Lane 2 : ab216144 IP in HeLa (CCCP-treated, ab141229) lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab216144 in HeLa (CCCP-treated, ab141229) whole cell lysate.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-PINK1 antibody [EPR20730] (ab216144)

Predicted band size: 63 kDa

Observed band size: 62 kDa

true

Exposure time: 5s

• WB

Lab

Western blot - Anti-PINK1 antibody [EPR20730] (AB216144)

Blocking and dilution buffer : 5% NFDM/TBST

PINK1 can be induced by CCCP treatment (PMID : 24184327).

All lanes:

Western blot - Anti-PINK1 antibody [EPR20730] (ab216144) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HeLa cells (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP, <a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 24 hours) 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

Predicted band size: 63 kDa

Observed band size: 62 kDa

true

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-PINK1 antibody [EPR20730] (AB216144)

Anti-PINK1 antibody [EPR20730] (ab216144) staining at 1/200 dilution shown in black; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. ab216144 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).  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 and incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent ab133456) 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.

All lanes:

Western blot - Anti-PINK1 antibody [EPR20730] (ab216144) at 1/200 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, 24h) 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

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 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

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 [EPR20730] (AB216144)

Dilution/blocking buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PINK1 antibody [EPR20730] (ab216144) at 1/1000 dilution

All lanes:

Human PINK1 recombinant protein (aa156-507), 10 ng

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 1/50000 dilution

Predicted band size: 63 kDa

Observed band size: 38 kDa

true

Exposure time: 1s