Leucine-Rich Repeat Kinase 2 (LRRK2) (C-Term), (AA 2500-2527) antibody

Details for Product No. ABIN152609
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Antigen
Synonyms LRRK2, AURA17, DARDARIN, PARK8, RIPK7, ROCO2, 4921513O20Rik, 9330188B09Rik, AW561911, D630001M17Rik, Gm927, cI-46
Epitope
C-Term, AA 2500-2527
(46), (38), (36), (19), (12), (12), (8), (6), (6), (6), (3), (3), (2), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1)
Reactivity
Cow (Bovine), Human, Mouse (Murine)
(175), (72), (69), (33), (13), (13), (2)
Host
Rabbit
(99), (80), (6), (4)
Clonality
Polyclonal
Conjugate
Un-conjugated
(11), (10), (8), (6), (6), (3), (3), (3), (3), (3), (3), (3), (3), (3), (3), (3), (3), (3), (3), (3), (2), (2), (2), (2), (2), (2), (2), (2), (2), (1)
Application
Western Blotting (WB), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP)
(161), (68), (43), (42), (33), (20), (16), (12), (12), (6), (2), (1), (1)
Pubmed 3 references available
Quantity 0.025 mL
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Catalog No. ABIN152609
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Immunogen A C-terminal synthetic peptide made to the human LRRK2 protein sequence (between residues 2500-2527). Immunogen sequence has 87% homology to rat
Purification affinity purified
Alternative Name PARK8 / LRRK2
Background LRRK2 is a 286 kDa protein belonging to the ROCO family. It is a multifunction proteininvolved in Parkinson's disease. Alternate Names: anti-LRRK-2 antibody, anti-LRRK 2 antibody, anti-PARK8 antibody, anti-ROCO2antibody, anti-Dardarin antibody, anti-BC 300-268 antibody.
Gene Symbol: LRRK2
Gene ID 120892
UniProt Q5S007
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.
Recommended dilutions: Immunocytochemistry/Immunofluorescence 1:1000-1:2000, Immunohistochemistry 1:500-1:1000, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin 1:500-1:1000, Immunoprecipitation 1:10-1:500, Western Blot 1:2000-1:5000
Protocol Protocol specific for LRRK2 Antibody Immunofluorescence Protocol:
1. Mouse CAD cells were transfected with Human wild-type LRRK-
.
. 48hr following transfection, the cells were fixed for 10 min with ice-cold MeOH.
. The cells were washed 3X with PBS, blocked with 10 % normal donkey serum (NDS) in PBS containing 0.25 % Triton for 1 hr at room temperature.
. The cells were incubated overnight at 4C with rabbit anti-LRRK2 (Cat. # NB 300-268) diluted 1/2000 in 1 % NDS in PBS containing 0.25 % Triton.
. The following day, the cells were washed 5X with PBS and incubated with FITC conjugated donkey anti-rabbit (1/100) diluted in 1 % NDS in PBS containing 0.25 % Triton for 1 hr.
. The cells were washed 5X with PBS and mounted with Vectashield, and visualized with a 40X oil-immersion objective.Protocol for immunoprecipitation of LRRK2 followed by LRRK2 autophosphorylation kinase assayCell lysis3X15 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. Preclear10 µg 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 µL 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 AbTo two of the tubes containing precleared lysate were added 7 µL 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 µL 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 µL) of buffer B for a total of 60ul of immunoprecipitate.Autophosphorylation kinase reaction, gel electrophoresis and phosphoimagingOn ice, 40 µL of immunoprecipitate from each tube was transferred to a .5ml kinase reaction tube. Each of the three reactions was supplemented with a 5 µL 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.
Restrictions For Research Use only
Format Liquid
Concentration 1 mg/mL
Buffer Tris-citrate/Phosphate, pH 7-8, Sodium Azide
Preservative Sodium azide
Precaution of Use WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.
Handling Advice Do not freeze.
Storage 4 °C
Supplier Images
anti-Leucine-Rich Repeat Kinase 2 (LRRK2) (C-Term), (AA 2500-2527) antibody Detection of LRRK2 in HeLa whole cell lysate (RIPA) using ABIN152608. 1:5,000 dilution, 1 minute ECL detection.
anti-Leucine-Rich Repeat Kinase 2 (LRRK2) (C-Term), (AA 2500-2527) antibody (2) Detection of LRRK2 in 50 ug of crude bovine brain membrane using ABIN152608.
anti-Leucine-Rich Repeat Kinase 2 (LRRK2) (C-Term), (AA 2500-2527) antibody (3) Mouse CAD cells transfected with Human wild-type LRRK-2 (1:2,000)
General Zimprich, Biskup, Leitner et al.: "Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology." in: Neuron, Vol. 44, Issue 4, pp. 601-7, 2004 (PubMed).

Miklossy, Arai, Guo et al.: "LRRK2 expression in normal and pathologic human brain and in human cell lines." in: Journal of neuropathology and experimental neurology, Vol. 65, Issue 10, pp. 953-63, 2006 (PubMed).

Melrose, Kent, Taylor et al.: "A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease." in: Neuroscience, Vol. 147, Issue 4, pp. 1047-58, 2007 (PubMed).

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