Anti-LRRK2 antibody [MJFF2 (c41-2)] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-LRRK2 antibody [MJFF2 (c41-2)] - BSA and Azide free (AB172378)

This data was developed using ab133474, the same antibody clone in a different buffer formulation.

LRRK2 was immunoprecipitated from 0.35 mg A549 (Human lung carcinoma epithelial cell) whole cell lysate 10 μg with 133474 at 1/60 dilution (2μg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab133474 IP in A549 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab133474 in A549 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Fresh lysate should be used to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-LRRK2 antibody [MJFF2 (c41-2)] (<a href='/ko/products/primary-antibodies/lrrk2-antibody-mjff2-c41-2-ab133474'>ab133474</a>)

Predicted band size: 286 kDa

Observed band size: 286 kDa

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• WB

Lab

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] - BSA and Azide free (AB172378)

This WB data was generated using the same anti-LRRK2 antibody clone, MJFF2 (c41-2), in a different buffer formulation (cat# ab133474).

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

Lanes 1 - 4 : Merged signal (red and green). Green - ab133474 observed at 286 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab133474 was shown to recognize LRRK2 in wild type A549 and MEF cells along with additional cross reative bands. Whilst signal was not seen in LRRK2 knockout cells. Wild-type and LRRK2 knockout samples were subjected to SDS-PAGE. ab133474 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 10000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed 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 a generous gift from Professor Dario Alessi, MRC Protein Phosphorylation and Ubiquitination Unit (University of Dundee).

All lanes:

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] (<a href='/ko/products/primary-antibodies/lrrk2-antibody-mjff2-c41-2-ab133474'>ab133474</a>)

Predicted band size: 286 kDa

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• WB

Lab

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] - BSA and Azide free (AB172378)

This data was developed using ab133474, the same antibody clone in a different buffer formulation.

ab133474 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 ab133474 overnight at 4 °C at a 1/10000 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 [MJFF2 (c41-2)] (<a href='/ko/products/primary-antibodies/lrrk2-antibody-mjff2-c41-2-ab133474'>ab133474</a>) at 1/10000 dilution

Lane 1:

Wild-type A549 lysate at 25 µg

Lane 2:

LRRK2 knock-out A549 lysate at 25 µg

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