Anti-LRRK2 抗体 [MJFF2 (c41-2)]

• WB

Lab

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] (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)] (ab133474)

Predicted band size: 286 kDa

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• IP

Lab

Immunoprecipitation - Anti-LRRK2 antibody [MJFF2 (c41-2)] (AB133474)

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)] (ab133474)

Predicted band size: 286 kDa

Observed band size: 286 kDa

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• IP

PubMed

Immunoprecipitation - Anti-LRRK2 antibody [MJFF2 (c41-2)] (AB133474)

Immunoprecipitation to verify the interaction of LRRK2 and ArfGAP1 in vivo. LRRK2 interacts with ArfGAP1 in brain extracts derived from wild-type mice following immunoprecipitation with ab133474, a LRRK2-specific monoclonal antibody (MJFF-2/c41-2), whereas ArfGAP1 is not immunoprecipitated in extracts derived from LRRK2 knockout mice

Protein extracts were prepared from the cerebral cortex of adult wild-type and LRRK2 knockout mice (with targeted deletion of exon 41 of the LRRK2 gene) by homogenization in TNE buffer (10 mM Tris-HCL pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1× phosphatase inhibitor cocktail 1 and 2, 1× Complete Mini protease inhibitor cocktail). Protein concentration was determined by BCA assay. Brain extracts (10 mg protein) were combined with 50 μl Protein G-Dynabeads pre-incubated with rabbit anti-LRRK2 (5 μg; MJFF2/c41-2; Abcam, Inc.), rabbit anti-ArfGAP1 (3 μg) or rabbit IgG (3 μg) antibodies followed by overnight incubation at 4°C. Dynabead complexes were sequentially washed twice with TNE buffer and twice with TBS buffer (10 mM Tris-HCL pH 7.4, 150 mM NaCl). Immunoprecipitates were eluted by heating at 70°C for 10 min, resolved by SDS-PAGE and subjected to Western blot analysis.

All lanes:

Immunoprecipitation - Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474)

Predicted band size: 286 kDa

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Stafa K. et al., PLoS Genet. 2012;8(2):e1002526. Fig 1 doi: 10.1371/journal.pgen.1002526. Reproduced under the Creative Commons license

• WB

Lab

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] (AB133474)

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)] (ab133474) 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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• WB

Supplier Data

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] (AB133474)

All lanes:

Western blot - Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474) at 1/10000 dilution

Lane 1:

HEK293 cell lysate transfected with 3*Flag vector at 10 µg

Lane 2:

HEK293 cell lysate transfected with 3*Flag full length wild type LRRK2 at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 286 kDa

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This image is courtesy of Zhuohua Zhang Lab (Sanford-Burnham Medical Research Institute)

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-LRRK2 antibody [MJFF2 (c41-2)] (AB133474)

Immunocytochemistry-immunofluorescence using Anti-LRRK2 antibody [MJFF2 (c41-2)], ab133474. Publication image from Bieri, G. et al., 2019, Acta Neuropathol, 30927072. Legend direct from paper.

The neuroinflammatory response is altered in PFF-injected LRRK2 G2019S mutant mice. a–c Representative images (a) and quantification (b, c) of microglial markers Iba1 (green) and Cd68 (red) in the dorsal striatum of LRRK2 G2019S (G2019S or GS) mice and wild-type (WT) littermate controls 6 months post injection (PI) with PFFs or Vehicle (Veh) control (n = 6–12 animals/group). d–f Experimental design and quantification of gene expression of microglial (e) and astrocyte-associated (f) activation markers. mRNA was isolated from the striatum and gene expression was analyzed using RT-qPCR analysis (n = 5–6 animals/group). g–h Representative images (g) and quantification (h) of intensity of C1q immunolabeling in the dorsal striatum of PFF and vehicle-injected mice 6 months post injection (n = 5–6 animals/group). Data expressed as mean + SEM; *p < 0.05, **p < 0.01; compared by Student’s t test, one-way ANOVA with a Tukey’s post-test for multiple comparisons or and two-way ANOVA with Bonferroni post hoc correction