Anti-PARK7/DJ1 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PARK7/DJ1 antibody (AB18257)

ab18257 staining PARK7/DJ1 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab18257 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PARK7/DJ1 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : Human brain tissue lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab18257 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab18257 was shown to specifically react with PARK/DJ1 in wild-type HAP1 cells. No band was observed when PARK/DJ1 knockout samples were used. Wild-type and PARK/DJ1 knockout samples were subjected to SDS-PAGE. ab18257 and ab8245 (loading control to GAPDH) were diluted to 1μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

All lanes:

Western blot - Anti-PARK7/DJ1 antibody (ab18257)

Predicted band size: 20 kDa

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• WB

Project

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

All lanes:

Western blot - Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/mL

Lane 1:

Brain (Mouse) Tissue Lysate at 10 µg

Lane 2:

Liver (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 3:

Heart (Mouse) Tissue Lysate at 10 µg

Lane 4:

Kidney (Mouse) Tissue Lysate at 10 µg

Lane 5:

Western blot - Mouse pancreas tissue lysate - total protein (<a href='/en-us/products/tissue-lysates/mouse-pancreas-tissue-lysate-total-protein-ab29363'>ab29363</a>) at 10 µg

Lane 6:

Testis (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 7:

Western blot - Mouse skeletal muscle tissue lysate - total protein (<a href='/en-us/products/tissue-lysates/mouse-skeletal-muscle-tissue-lysate-total-protein-ab29711'>ab29711</a>) at 10 µg

Lane 8:

Spinal Cord (Mouse) Tissue Lysate at 10 µg

Lane 9:

Ovary (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 10:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Lane 11:

Brain (Rat) Tissue Lysate - normal tissue at 10 µg

Lane 12:

Liver (Rat) Tissue Lysate at 10 µg

Lane 13:

Heart (Rat) Tissue Lysate at 10 µg

Lane 14:

Kidney (Rat) Whole Cell Lysate - normal tissue (<a href='/en-us/products/unavailable/kidney-rat-whole-cell-lysate-normal-tissue-ab29480'>ab29480</a>) at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 20 kDa

Observed band size: 15 kDa,24 kDa

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• WB

Unknown

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

All lanes:

Western blot - Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/mL

All lanes:

Western blot - Recombinant Human PARK7/DJ1 protein (His tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-park7-dj1-protein-ab51198'>ab51198</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 20 kDa

true

Exposure time: 1min

• WB

Unknown

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

All lanes:

Western blot - Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/mL

All lanes:

Western blot - Recombinant Human PARK7/DJ1 protein (His tag N-Terminus) (<a href='/en-us/products/proteins-peptides/recombinant-human-park7-dj1-protein-ab51198'>ab51198</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 20 kDa

true

Exposure time: 1min

• WB

Project

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

All lanes:

Western blot - Anti-PARK7/DJ1 antibody (ab18257) at 1 µg/mL

Lane 1:

Jurkat lysate at 20 µg

Lane 2:

HeLa lysate at 20 µg

Lane 3:

3T3 lysate at 20 µg

Lane 4:

Jurkat lysate at 20 µg with Human PARK7/DJ1 peptide (<a href='/en-us/products/proteins-peptides/human-park7-dj1-peptide-ab18659'>ab18659</a>)

Lane 5:

HeLa lysate at 20 µg with Human PARK7/DJ1 peptide (<a href='/en-us/products/proteins-peptides/human-park7-dj1-peptide-ab18659'>ab18659</a>)

Lane 6:

3T3 lysate at 20 µg with Human PARK7/DJ1 peptide (<a href='/en-us/products/proteins-peptides/human-park7-dj1-peptide-ab18659'>ab18659</a>)

Secondary

All lanes:

Alexa Fluor Goat polyclonal to Rabbit IgG (680) at 1/10000 dilution

Predicted band size: 20 kDa

Observed band size: 24 kDa

false

• WB

CiteAb

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

Western Blotting using Anti-PARK7/DJ1 antibody, ab18257. Publication image from Zhang, Y. et al., 2011, Mol Neurodegener, 21645326. Legend direct from paper.

Loss of function of DJ-1 increased the aggregation of MAP1b-LC. A, DJ-1 shRNA vector or scramble shRNA vector was transfected into SH-SY5Y cells and selected with 300 µg hygromycin. The stable clone was picked and amplified. Western blot result showed efficient down-regulation of DJ-1. B, Flag-MAP1b-LC was transfected into DJ-1 shRNA or scramble SH-SY5Y cells for 48 hrs. Cells were lysed and separated into Triton X-100 soluble or insoluble components. The result showed increased insoluble MAP1b-LC in the DJ-1 KD cells. C, Flag-MAP1b-LC was transfected into DJ-1 shRNA SH-SY5Y or scramble shRNA cells for 48 hrs. Cells were immuno-stained with rabbit anti-Flag antibody. The results showed the enhancement of MAP1b-LC formed aggregates in DJ-1 KD cells. D, The total lysates of DJ-1 KD SH-SY5Y cells or scramble controls were separated into Triton X-100 soluble or insoluble fractions. Increased insoluble MAP1b-LC in DJ-1 KD cells was observed by Western blot. E, Endogenous MAP1b-LC was examined by immunofluorescence assay in DJ-1 KD SH-SY5Y cells or scramble controls. The MAP1b-LC formed aggregates increased in DJ-1 KD cells. F and G, DJ-1 deficiency intensified the formation of insoluble MAP1b-LC in vivo. F, The wild type or DJ-1 KO mice brain lysates were separated into Triton X-100 soluble or insoluble fractions. Western blot was used to analysis of MAP1b-LC distribution in the soluble or insoluble fractions. G, Quantification of relative levels of MAP1b-LC (*, p = 0.03).

false

• WB

CiteAb

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

Western Blotting using Anti-PARK7/DJ1 antibody, ab18257. Publication image from Zhang, Y. et al., 2011, Mol Neurodegener, 21645326. Legend direct from paper.

Loss of function of DJ-1 increased the aggregation of MAP1b-LC. A, DJ-1 shRNA vector or scramble shRNA vector was transfected into SH-SY5Y cells and selected with 300 µg hygromycin. The stable clone was picked and amplified. Western blot result showed efficient down-regulation of DJ-1. B, Flag-MAP1b-LC was transfected into DJ-1 shRNA or scramble SH-SY5Y cells for 48 hrs. Cells were lysed and separated into Triton X-100 soluble or insoluble components. The result showed increased insoluble MAP1b-LC in the DJ-1 KD cells. C, Flag-MAP1b-LC was transfected into DJ-1 shRNA SH-SY5Y or scramble shRNA cells for 48 hrs. Cells were immuno-stained with rabbit anti-Flag antibody. The results showed the enhancement of MAP1b-LC formed aggregates in DJ-1 KD cells. D, The total lysates of DJ-1 KD SH-SY5Y cells or scramble controls were separated into Triton X-100 soluble or insoluble fractions. Increased insoluble MAP1b-LC in DJ-1 KD cells was observed by Western blot. E, Endogenous MAP1b-LC was examined by immunofluorescence assay in DJ-1 KD SH-SY5Y cells or scramble controls. The MAP1b-LC formed aggregates increased in DJ-1 KD cells. F and G, DJ-1 deficiency intensified the formation of insoluble MAP1b-LC in vivo. F, The wild type or DJ-1 KO mice brain lysates were separated into Triton X-100 soluble or insoluble fractions. Western blot was used to analysis of MAP1b-LC distribution in the soluble or insoluble fractions. G, Quantification of relative levels of MAP1b-LC (*, p = 0.03).

false

• WB

CiteAb

Western blot - Anti-PARK7/DJ1 antibody (AB18257)

Western Blotting using Anti-PARK7/DJ1 antibody, ab18257. Publication image from Zhang, Y. et al., 2011, Mol Neurodegener, 21645326. Legend direct from paper.

DJ-1 interacted with MAP1b light chain. A, GST-DJ-1 or GST was incubated with 6xhis-MAP1b-LC for 3 hrs and then was pulled down by GST beads, the beads were washed and SDS-PAGE followed by Coomassie blue staining was used to analyze the result. The result showed DJ-1 could bind to MAP1b-LC directly in vitro. B, Flag-MAP1b-LC and HA-DJ-1 were transfected into HEK293t cells for 36 hrs and the cell lysates were immunoprecipitated with Flag M2 beads or HA antibody conjugated beads. Both DJ-1 and MAP1b-LC were detected by western blot with HA or Flag antibody respectively. C and D, Co-localization of MAP1b-LC and DJ-1. Flag-MAP1b-LC and HA-DJ-1 were cotransfected into KEK293t (C) or SH-SY5Y cells (D) for 36 hrs before fixed and stained with rabbit anti-Flag and mouse anti-HA antibodies. E. The wild type or DJ-1 KO mouse brain lysates were immunoprecipitated with DJ-1 antibody, MAP1b-LC could be detected in the wild type mouse brain but not in that of the KO mouse. F, The primary cortical neuron was fixed and immuno-stained with DJ-1 and MAP1b-LC antibody.

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