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Products:Neuroscience >> Cell Type Marker >> Neuron marker >> Synapse marker
Overview
Product name
Anti-Piccolo antibody - Synaptic Marker
See all Piccolo products (6) ...
Description
Rabbit polyclonal to Piccolo - Synaptic Marker
Specificity
Due to its large size, Piccolo requires special gel-electrophoresis and Western blotting protocols for visualization by immunoblotting. Excellent results can be obtained, for example, with the 4-12% TRIS-glycine gradient gels of Anamed. For success in WB with Rabbit polyclonal to Piccolo - Synaptic Marker (ab20664), do not denature WB sample lysate.
Tested applications
IHC-Fr, ICC, IHC (PFA fixed), WB, IHC-Pmore details
Cross reactivity
Reacts with
Mouse, Rat
Immunogen
Synthetic peptide conjugated to KLH derived from within residues 600 - 700 of Rat Piccolo.
(Peptide available as ab23400.)
Positive control
Rat brain and mouse brain
Properties
Form
Liquid
Storage instructions
Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
Storage buffer
Preservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
Concentration
Concentration information loading...
Purity
Immunogen affinity purified
Clonality
Polyclonal
Isotype
IgG
Research areas
Neuroscience >> Cell Type Marker >> Neuron marker >> Synapse marker
Applications
Show applications key
Our Abpromise guarantee covers the use of ab20664 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
IHC-Fr: 1/500(PubMed: 19218615)
ICC: Use at an assay dependent dilution. (PubMed: 19377471)
IHC (PFA fixed): 1/300 - 1/1000.
WB: Use a concentration of 0.2 - 0.5 µg/ml.Detects a band of approximately 520 kDa (predicted molecular weight: 520 kDa).Can be blocked with Piccolo peptide (ab23400).
IHC-P: Use a concentration of 5 µg/mlPerform heat mediated antigen retrieval before commencing with IHC staining protocol.
Target
Function
May act as a scaffolding protein involved in the organization of synaptic active zones and in synaptic vesicle trafficking.
Sequence similarities
Contains 2 C2 domains.
Contains 1 PDZ (DHR) domain.
Domain
C2 domain 1 is involved in binding calcium and phospholipids. Calcium binds with low affinity but with high specificity and induces a large conformational change.
Cellular localization
Cell junction > synapse. Concentrated at the presynaptic side of synaptic junctions.
Target information above from: UniProt accessionQ9Y6V0
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010).
Alternative names
- ACZ antibody
- Aczonin antibody
- Brain-derived HLMN protein antibody
see all
Database links
- Entrez Gene: 26875 Mouse
- Entrez Gene: 56768 Rat
- SwissProt: Q8CF91 Mouse
- SwissProt: Q8CF92 Mouse
- SwissProt: Q9QYX7 Mouse
- SwissProt: Q9JKS6 Rat
- Unigene: 146275 Mouse
- Unigene: 53925 Rat
Anti-Piccolo antibody - Synaptic Marker images:
Western blot - Piccolo antibody - Synaptic Marker (ab20664)
All lanes : Anti-Piccolo antibody - Synaptic Marker (ab20664) at 0.2 µg/ml
Lane 1 : Rat brain lysate at 40 µg
Lane 2 : Mouse brain lysate at 50 µg
Secondary
Anti-rabbit HRP at 1/20000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 520 kDa
Observed band size : 520 kDa
Additional bands at : 60 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 10 minutes
Primary Antibody : anti-Piccolo (ab20664) for 1h in 5% Whole Milk Powder (WMP); Secondary Antibody : Anti-rabbit HRP (1/20000) for 1h in 5% WMP; Migration medium: Laemmeli + glycerol + 5% B-mercaptoethanol; Transfer : Tris/Glycine, 20% ethanol
NB: Gels higher than 8% acrylamide were tried without success; 8% or lower is recommended. Denaturing of samples at 70C or 95C was not successful. Reducing conditions were used and 20% ethanol was employed for the nitrocellulose membrane transfer. All steps performed at RT.
Randal Moldrich, CNRS UMR7637, ESPCI, France
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Piccolo antibody - Synaptic Marker (ab20664)
IHC image of Piccolo staining in rat brain FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20664, 5µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
Western blot - Piccolo antibody - Synaptic Marker (ab20664)
All lanes : Anti-Piccolo antibody - Synaptic Marker (ab20664) at 1 µg/ml
Lane 1 : Brain (Rat) Tissue Lysate
Lane 2 : Brain (Mouse) Tissue Lysate
Lane 3 : Brain (Rat) Tissue Lysate with Piccolo peptide (ab23400) at 1 µg/ml
Lane 4 : Brain (Mouse) Tissue Lysate with Piccolo peptide (ab23400) at 1 µg/ml
Lysates/proteins at 30 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 520 kDa
Observed band size : 275 kDa (why is the actual band size different from the predicted?)
Additional bands at : 31 kDa,60 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 4 minutes
Immunohistochemistry (PFA fixed) - Piccolo antibody - Synaptic Marker (ab20664)
Immuofluorescent staining for Piccolo ab20664 in [A] rat brain hippocampus (X20 objective) and [B] rat brain cortex (X40 objective). Tissue preparation: rat brain tissue was perfusion fixed (4% PFA) followed by post fix and cryoprotection in 20% sucrose before freezing in OCT. 30µm coronal sections were cut on a cryostat for free floating IHC. Primary antibody ab20664 was used at 1/100 (5µg/ml) incubated overnight at room temperature in PBST (triton 0.3%). Secondary antibody used: anti-rabbit Alexa fluor 488 (1/1000) incubated for 2 hours at room temperature.
Sophie Pezet, King`s college London, United Kingdom
References for Anti-Piccolo antibody - Synaptic Marker (ab20664)
This product has been referenced in:
- Konishi Y & Setou M Tubulin tyrosination navigates the kinesin-1 motor domain to axons. Nat Neurosci 12:559-67 (2009). ICC; Rat.Read more (PubMed: 19377471) »
- Grossman GHet al. Early synaptic defects in tulp1-/- mice. Invest Ophthalmol Vis Sci 50:3074-83 (2009). IHC-Fr; Rat.Read more (PubMed: 19218615) »
See all 4 publications for this product
Publishing research using ab20664? Please let us know so that we can cite the reference in this datasheet
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"