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Key Product Details

Validated by

Knockout/Knockdown, Independent Antibodies

Species Reactivity

Human, Mouse, Bovine, C. elegans, Insect, Plant

Applications

Flow (Intracellular), Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockout Validated, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1 mg/ml

Product Summary for LRRK2 Antibody - BSA Free

Immunogen

A C-terminal synthetic peptide made to the human LRRK2 protein sequence (between residues 2500-2527). [UniProt# Q5S007]

Reactivity Notes

Human, Moth reactivity reported in scientific literature (PMID: 19824698). Plant reactivity reported in scientific literature (PMID: 27273569).

Localization

Mitochondrial outer membrane.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

286 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for LRRK2 Antibody - BSA Free

Western Blot: LRRK2 Antibody [NB300-268] - Western blotting of LRRK2. Recombinant LRRK2 (arrowhead) from transfected (+) HEK 293T and M17 cells was specifically recognized by all four LRRK2 antibodies (Ab1, Ab2, Ab3, and Ab4) used in this study. LRRK2 was not recognized in non-transfected cells (-). Image collected and cropped by CiteAb from the following publication (http://molecularneurodegeneration.biomedcentral.com/articles/10.1186/1750-1326-1-17), licensed under a CC-BY license.
Immunohistochemistry-Frozen: LRRK2 Antibody [NB300-268] - Staining as described in PMID 24312256. Image from verified customer review.
Knockout Validated: LRRK2 Antibody [NB300-268] - Mn increases LRRK2 kinase activity and expression in RAW 264.7 and HMC3 cells. Two-hundred (200) ug of protein were collected from cell lysates, followed by western blot analysis to determine the presence of LRRK2 in LRRK2 WT, KO RAW 264.7 and HMC3 cells. Beta-actin was used as a loading control. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0210248) licensed under a CC-BY license.

Applications for LRRK2 Antibody - BSA Free

Application
Recommended Usage

Flow (Intracellular)

2.5 ug/ml

Flow Cytometry

2.5 ug/ml

Immunocytochemistry/ Immunofluorescence

2 - 5 ug/ml

Immunohistochemistry

1:200

Immunohistochemistry-Paraffin

1:200

Immunoprecipitation

1:10-1:500

Western Blot

1 - 2 ug/ml
Application Notes
In Western blot, a band can be seen at ~286 kDa. We have also seen other bands with some lysates, but these bands have been blocked by the control peptide, suggesting that these bands are degradation products. IP has been done in an LRRK2 autophosphorylation kinase assay, IHC has been done on brain sections and ICC/IF has been done on transfected cell lines. Frozen sections using the LRRK2 antibody were from a customer review. The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 1 review rated 5 using NB300-268 in the following applications:

Published Applications

Read 46 publications using NB300-268 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.1% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Store at -20C long term. Avoid freeze-thaw cycles.

Background: LRRK2

LRRK2 (leucine-rich repeat serine/threonine-protein kinase 2, also called PARK8 or Dardarin) is a homodimeric multifunctional protein that belongs to TKL serine threonine protein kinases family and LRRK2 immunoreactivity has been documented in neurofibrillary tangles in Alzheimer's disease as well as in PDCG (parkinsonism-dementia complex of Guam). Highly expressed in tissues such as liver, heart, brain etc., LRRK2 localizes to cellular cytoplasm and also associates with membrane structures including mitochondrial outer membrane. LRRK interacts with PARK2, PRDX3 and TPCN2, and phosphorylate proteins which are implicated in Parkinson disease. LRRK2 is a cytoplasmic kinase with autophosphorylation capability as well as GTPase activity. Its interaction with microtubules suggests that LRRK2-induced neurodegeneration might be partly mediated via inhibition of microtubule dynamics, and LRRK2 -mitochondria interactiosn suggests its implication in pathways eliciting ROS mediated damage. LRRK2 has been shown exert positive regulation in autophagy via calcium-sensitive activation of CaMKK/AMPK signaling pathway through activation of NAADP receptors, increase of lysosomal pH as well as calcium release.

Long Name

Leucine-rich Repeat Ser/Thr Protein Kinase 2

Alternate Names

AURA17, Dardarin, PARK8, ROCO2

Entrez Gene IDs

120892 (Human); 66725 (Mouse)

Gene Symbol

LRRK2

UniProt

Product Documents for LRRK2 Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for LRRK2 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for LRRK2 Antibody - BSA Free (NB300-268):

[[URL:https://www.novusbio.com/products/lrrk2-antibody_nb300-268]][[Caption:L… Antibody]]
IHC-FFPE sections
I. Deparaffinization:
A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
B. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.

II. Quench Endogenous Peroxidase:
A. Place slides in peroxidase quenching solution: 15-30 minutes.
To Prepare 200 ml of Quenching Solution:
Add 3 ml of 30% Hydrogen Peroxide to 200 ml of Methanol.
Use within 4 hours of preparation
B. Place slides in distilled water: 2 changes for 2 minutes each.

III. Retrieve Epitopes:
A. Preheat Citrate Buffer. Place 200 ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees Celcius.
B. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
C. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
D. Slowly add distilled water to further cool for 5 minutes.
E. Rinse slides with distilled water. 2 changes for 2 minutes each.

IV. Immunostaining Procedure:
A. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap Pen).
B. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.
C. Drain wash solution and apply 4 drops of Blocking Reagent to each slide and incubate for 15 minutes.
D. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200 ul of Primary Antibody solution to each slide, and incubate for 1 hour.
E. Wash slides with Wash Solution: 3 changes for 5 minutes each.
F. Drain wash solution, apply 4 drops of Secondary antibody to each slide and incubate for 1 hour.
G. Wash slides with Wash Solution: 3 changes for 5 minutes each.
H. Drain wash solution, apply 4 drops of DAB Substrate to each slide and develop for 5-10 minutes. Check development with microscope.
I. Wash slides with Wash Solution: 3 changes for 5 minutes each.
J. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
K. Wash slides with Wash Solution: 2-3 changes for 2 minutes each.
L. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
M. Rinse slides in distilled water.
N. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
O. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
P. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
Q. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
R. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.
S. Lay slides on a flat surface to dry prior to viewing under microscope.

NOTES:
-Use treated slides (e.g. HistoBond) to assure adherence of FFPE sections to slide.
-Prior to deparaffinization, heat slides overnight in a 60 degrees Celcius oven.
-All steps in which Xylene is used should be performed in a fume hood.
-For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.
-For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.
-200 ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections less than 200 ul may be used.
-5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.
-Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-1 1/2 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired increase time (up to 10 minutes).
[[URL:https://www.novusbio.com/products/lrrk2-antibody_nb300-268]][[Caption:L… Antibody ]]
Protocol for immunoprecipitation of LRRK2 followed by LRRK2 autophosphorylation kinase assay

Cell lysis
3X15 cm plates of SH-SY5Y cells were grown to 80% confluency. The plates were washed twice with PBS and placed on ice. Remaining PBS was aspirated off after tilting plate to remove all PBS. 1.5 ml of cold lysis buffer (buffer A) was added to each plate. The plates were allowed to incubate on ice 5 min until the cells detached. The lysis buffer and cells for each plate were then vigorously passed 6X through a 30.5 guage needle. Lysis buffer and cells were transferred to 3X1.5 ml eppendorf tubes and spun 5 min. at 5,000 rpm in a 4 degree eppendorf microfuge. Lysates were removed from pelleted debris and transferred to new 1.5 ml eppendorf tubes and recentrifuged 10 min. at 13,000 rpm. Lysate was transferred to three new tubes and 1/2 lysate volume of buffer A (-) NaCl was added to each tube.
Preclear
10 ug of rabbit IgG were added to the lysate for each tube and the lysate was vortexed followed by rotating at 4 degrees for 2 hours. 20 ul of protein A sepharose beads (Amersham cat#: 17-0469-01) were added. The tubes were vortexed and then rotated for 1.5 hours at 4 degrees. Lysates were separated from protein A beads beads by low (200 rpm) spin for 2 min and transferred to new eppendorf tubes. A repeat of the protein A sepharose incubation was carried out to remove residual rabbit IgG followed by removal of the protein A beads.

Immunoprecipitation with LRRK2 Ab

To two of the tubes containing precleared lysate were added 7 ul of LRRK2 Ab (7ug). To the remaining tube was added 7ug of rabbit IgG. The tubes were vortexed and allowed to rotate overnight at 4 degrees. The following morning 30 ul of protein A sepharose was added to each tube, the tubes were vortexed and rotated at 4 degrees for 2 hours. The protein A beads were then isolated by brief, low speed centrifugation and were washed 3X in 500ul buffer A (-) NaCl. This was followed by two washes in kinase buffer (buffer B). Protein A beads were resuspended in 1 volume (30 ul) of buffer B for a total of 60ul of immunoprecipitate.
Autophosphorylation kinase reaction, gel electrophoresis and phosphoimaging
On ice, 40 ul of immunoprecipitate from each tube was transferred to a .5ml kinase reaction tube. Each of the three reactions was supplemented with a 5 ul mixture that gave a final reaction concentration of 15 uM cold ATP and 5uCi ATP. The reaction mixtures were vortexed and transferred to a rotator in a 30 degree incubator. The autophosphorylation incubation was allowed to go for 30 minutes and the reaction tubes were taken off the rotator and vortexed every five minutes. The reactions were then halted by addition of 11ul of 5X SDS gel running sample buffer to each of the three samples. 40ul of each sample was then run on a 7% acrylamide-acetate mini-gel. Once the 200Kd molecular weight marker band had run half way down the gel, the gel was stopped dried and exposed blanked to a phosphoimaging cassette (Molecular Dynamics). Following 24 hour exposure, the cassette was assessed for radioactivity on a Storm analyzer.

Buffers

Buffer A (make 10ml both - and + NaCl solutions) = lysis buffer
50mM Tris pH 7.4
150mM NaCl
0.2% NP40
Protease inhibitor cocktail (stock = 100X, Sigma)
0.5mM vanadate
15mM EDTA
adjust to 10 ml with H2O
Buffer B = kinase buffer
10mM hepes
10mM MgCl2
50mM NaCl
protease inhibitor
vanadate
(1mM NaN3 if storing overnight or longer)

FAQs for LRRK2 Antibody - BSA Free

Showing  1 - 2 of 2 FAQs Showing All
  • Q: I am wondering what concentration of Lrrk2 blocking peptide (NB300-268PEP) I should use for FACS as a control?

    A: We do have a protocol for how to incubate the primary with the blocking peptide to perform a negative control competition assay. While this was written with WB and IHC in mind, it should work the same in inhibiting cell binding for flow.

  • Q: Is the immunogen of NB300-268 within amino acids 922 and 1143 of LRRK2 (Uniprot ID#Q5S007)?

    A: The immunogen used to generate NB300-268 was a synthetic peptide made to a C-terminal region within residues 2500-2527 of the human LRRK2 protein (UniProt# Q5S007). We do have an alternative antibody that you may be interested in, with catalog number NB110-55289. The immunogen used to generate this product was synthetic peptide made to an internal region of the mouse LRRK2 protein sequence (between residues 900-1000), although this does not recognize human LRRK2. If you are looking to detect mouse LRRK2 this would be a suitable product for you.

Showing  1 - 2 of 2 FAQs Showing All
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