Anti-LRRK2 antibody [UDD3 30(12)]

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

Western blot : Rabbit Monoclonal[UDD3 30(12)] to LRRK2 ab133518 staining at 1/1000 dilution, shown in green; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 268 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in LRRK2 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (ab133518) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human LRRK2 knockout U-87 MG cell line (ab306722) at 20 µg

Lane 3:

A549 at 20 µg

Lane 4:

HT-29 at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 286 kDa

Observed band size: 268 kDa,80 kDa

false

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

ab133518 was shown to react with LRRK2 in wild-type A549 cells in Western blot with loss of signal observed in a LRRK2 knockout cell line. Wild-type A549 and LRRK2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab133518 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were 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-LRRK2 antibody [UDD3 30(12)] (ab133518) at 1/500 dilution

Lane 1:

Wild-type A549 lysate at 25 µg

Lane 2:

LRRK2 knock-out A549 lysate at 25 µg

false

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

Immunofluorescent staining of SH-SY5Y cells fixed and permeablized with 4% PFA and 0.1% Triton X 100 using purified ab133518 at a dilution of 1/200. An Alexa Fluor^® 488 goat anti-rabbit was used as the secondary (ab150077, 1/400) and the sample was stained with DAPI. The negative control is shown in bottom right hand panel - for the negative control, purified ab133518 was used at a dilution of 1/200 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500.

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (ab133518) at 1/10000 dilution

All lanes:

LRRK2 over-expressed 293 cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 286 kDa

Observed band size: 286 kDa

false

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (ab133518) at 1/1000 dilution

Lane 1:

NIH/3T3 cell lysate at 20 µg

Lane 2:

RAW264.7 cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 286 kDa

Observed band size: 286 kDa

false

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

Lane 1 : Wild-type A549 cell lysate (20 μg)
Lane 2 : LRRK2 knockout A549 cell lysate (20 μg)
Lane 3 : Wild-type MEF cell lysate (20 μg)
Lane 4 : LRRK2 knockout MEF cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab133518 observed at 238 kDa. Red - loading control, ab130007, observed at 124 kDa.

ab133518 was shown to specifically react with wild type A549 and MEF cell lines. No band was observed when knock out samples were used. Wild-type and LRRK2 knockout samples were subjected to SDS-PAGE. ab133518 and ab130007 (loading control to Vinculin) were diluted at 1/500 and 1/10000 dilution respectively and incubated overnight at 4C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

Wild-type and LRRK2 knockout MEF and A549 cells were provide as generous a gift from Professor Dario Alessi, MRC Protein Phosphorylation and Ubiquitination Unit (University of Dundee).

All lanes:

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (ab133518)

Predicted band size: 286 kDa

false

• WB

Unknown

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

All lanes:

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (ab133518) at 1/1000 dilution

Lane 1:

GFP-LRRK2 lysate at 5 µg

Lane 2:

GFP LRRK2 S910A lysate at 5 µg

Lane 3:

GFP LRRK2 S935A lysate at 5 µg

Lane 4:

LRRK2 WT MEF lysate at 20 µg

Lane 5:

LRRK2 WT MEF lysate from LRRK2-IN1 treated cells at 20 µg

Lane 6:

LRRK2 KO MEF lysate at 20 µg

Lane 7:

LRRK2 KO MEF lysate from LRRK2-IN1 treated cells at 20 µg

Lane 8:

Lymphoblastoid lysate at 30 µg

Lane 9:

Lymphoblastoid lysate from LRRK2-IN1 treated cells at 30 µg

Secondary

All lanes:

Goat-anti-rabbit HRP at 1/2000 dilution

Predicted band size: 286 kDa

false

• WB

CiteAb

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] (AB133518)

LRRK2 western blot using anti-LRRK2 antibody [UDD3 30(12)] ab133518. Publication image and figure legend from Antoniou, N., Vlachakis, D., et al., 2018, Sci Rep, PubMed 29472595.

ab133518 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab133518 please see the product overview.

Deletion of predicted FADD binding domain in LRRK2 ARM region is neuroprotective. Primary rat embryonic cortical neurons were transiently transfected with full-length mutant, or its corresponding deletion pair (ΔFBD), together with a construct encoding an EGFP reporter at a ratio of 4 : 1. Following 72 h of expression, neurons were fixed and processed for anti-LRRK2/EGFP immunofluorescence with DAPI nuclear stain (a). Please see Fig. 2 and Supplementary Fig. 11 for representative images of full-length mutant LRRK2. Panel (b) shows the relative expression levels of full-length or deletion constructs. HEK293T cells were transiently transfected with full-length mutant LRRK2, or mutant LRRK2 (ΔFBD) lacking the FADD binding domain. Cell extracts were separated by SDS-PAGE and the membranes probed with anti-LRRK2 (c41-2) and β-actin, as a loading control. In (c), the percentage of EGFP-positive neurons displaying apoptotic nuclear morphology was determined by a rater blinded to the experimental conditions. **p < 0.01 compared to full-length mutant LRRK2; ***p < 0.001 compared to full-length mutant LRRK2. Each data point represents the mean +/- SEM from 3-4 independent transfections. Cultures were repeated at least 3 times with similar differences.

false