Anti-LRRK2 antibody [UDD3 30(12)] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LRRK2 antibody [UDD3 30(12)] - BSA and Azide free (AB170993)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133518).

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] - BSA and Azide free (AB170993)

This data was developed using ab133518, the same antibody clone in a different buffer formulation.

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)] (<a href='/en-us/products/primary-antibodies/lrrk2-antibody-udd3-3012-ab133518'>ab133518</a>) 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)] - BSA and Azide free (AB170993)

This data was developed using ab133518, the same antibody clone in a different buffer formulation.

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)] (<a href='/en-us/products/primary-antibodies/lrrk2-antibody-udd3-3012-ab133518'>ab133518</a>) at 1/500 dilution

Lane 1:

Wild-type A549 lysate at 25 µg

Lane 2:

LRRK2 knock-out A549 lysate at 25 µg

false

• WB

Lab

Western blot - Anti-LRRK2 antibody [UDD3 30(12)] - BSA and Azide free (AB170993)

This WB data was generated using the same anti-LRRK2 antibody clone, UDD3 30(12), in a different buffer formulation (cat# 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)] (<a href='/en-us/products/primary-antibodies/lrrk2-antibody-udd3-3012-ab133518'>ab133518</a>)

Predicted band size: 286 kDa

false