Anti-PARK7/DJ1 antibody [EPR19466-105]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed HeLa (human cervix adenocarcinoma) cell line labeling PARK7/DJ1 with ab201147 at 1/120 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluorr^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

ab201147 was shown to react with PARK7 in wild-type HEK-293T cells in immunocytochemistry with loss of signal observed in PARK7 knockout cell line ab266338. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab201147 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

Immunofluorescent analysis of 100% methanol fixed LNCaP (Human prostate cancer cell line) cells labeling PARK7/DJ1 with ab201147 at 1/100 dilution, followed by AlexaFluo^r®488 Goat Anti-Rabbit (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and weak nuclear staining on LNCaP cells.

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).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

Immunofluorescent analysis of 100% methanol fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling PARK7/DJ1 with ab201147 at 1/100 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and weak nuclear staining on HeLa cells.

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).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

PARK7/DJ1 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) whole cell lysate with ab201147 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab201147 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1 : HEK-293T (human embryonic kidney) whole cell lysate 10 μg (Input).
Lane 2 : HEK-293T whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab201147 in HEK-293T whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-PARK7/DJ1 antibody [EPR19466-105] (ab201147)

Predicted band size: 20 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (ab201147) at 1/5000 dilution

Lane 1:

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

Lane 2:

HEK-293T (human embryonic kidney) whole cell lysate at 20 µg

Lane 3:

LNCaP (human prostate carcinoma) 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 1/100000 dilution

Predicted band size: 20 kDa

Observed band size: 23 kDa

false

Exposure time: 5s

• WB

Collaborator

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

ab201147 was shown to react with PARK7 in wild-type HEK-293T cells in Western blot with loss of signal observed in PARK7 knockout cell line ab266338. Wild-type HEK-293T and PARK7 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab201147 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (ab201147) at 1/5000 dilution

Lane 1:

Wild-type HEK-293T lysate at 30 µg

Lane 2:

PARK7 knock-out HEK-293T lysate at 30 µg

Lane 2:

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (ab201147) at 30 µg

false

• WB

Lab

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (AB201147)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PARK7/DJ1 antibody [EPR19466-105] (ab201147) at 1/5000 dilution

Lane 1:

Human brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 20 kDa

Observed band size: 23 kDa

false

Exposure time: 5s