Anti-Piccolo antibody (ab20664)
Key features and details
- Rabbit polyclonal to Piccolo
- Suitable for: IHC (PFA fixed), WB, IHC-P
- Reacts with: Mouse, Rat, Human
- Isotype: IgG
Overview
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Product name
Anti-Piccolo antibody
See all Piccolo primary antibodies -
Description
Rabbit polyclonal to Piccolo -
Host species
Rabbit -
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
Suitable for: IHC (PFA fixed), WB, IHC-Pmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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General notes
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
Applications
The Abpromise guarantee
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.
Application Abreviews Notes IHC (PFA fixed) 1/300 - 1/1000.WB (1) Use a concentration of 0.2 - 0.5 µg/ml. Detects a band of approximately 460 kDa (predicted molecular weight: 520 kDa).Abcam recommends using milk as the blocking agent.
IHC-P (1) Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.Notes IHC (PFA fixed)
1/300 - 1/1000.WB
Use a concentration of 0.2 - 0.5 µg/ml. Detects a band of approximately 460 kDa (predicted molecular weight: 520 kDa).Abcam recommends using milk as the blocking agent.
IHC-P
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.Target
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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. - Information by UniProt
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Database links
- Entrez Gene: 27445 Human
- Entrez Gene: 26875 Mouse
- Entrez Gene: 56768 Rat
- Omim: 604918 Human
- SwissProt: Q9Y6V0 Human
- SwissProt: Q9QYX7 Mouse
- SwissProt: Q9JKS6 Rat
- Unigene: 12376 Human
see all -
Alternative names
- ACZ antibody
- Aczonin antibody
- Brain-derived HLMN protein antibody
see all
Images
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Piccolo antibody (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.
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Western blot - Anti-Piccolo antibody (ab20664)All lanes : Anti-Piccolo antibody (ab20664) at 1 µg/ml
Lane 1 : Brain (Rat) Tissue Lysate
Lane 2 : Cerebellum (Rat) Tissue Lysate
Lane 3 : Kidney (Rat) Tissue Lysate (Negative control)
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 520 kDa
Observed band size: 460 kDa why is the actual band size different from the predicted?
Additional bands at: 200 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 4 minutesThe banding pattern shown in the image above is consistent with the literature which describes multiple bands >420 kDa as a result of proteolysis (PMID : 10707984).
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes using lysates heated to 85°C before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab20664 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
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Immunohistochemistry (PFA fixed) - Anti-Piccolo antibody (ab20664)This image is courtesy of Sophie Pezet, King's College London, United KingdomImmuofluorescent 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. -
Western blot - Anti-Piccolo antibody (ab20664)This image is courtesy of Randal Moldrich, CNRS UMR7637, ESPCI, FranceAll lanes : Anti-Piccolo antibody (ab20664) at 0.2 µg/ml
Lane 1 : Rat brain lysate at 40 µg
Lane 2 : Mouse brain lysate at 50 µg
Secondary
All lanes : 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 minutesPrimary 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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (12)
ab20664 has been referenced in 12 publications.
- Valdivia AO & Bhattacharya SK Lyso-Lipid-Induced Oligodendrocyte Maturation Underlies Restoration of Optic Nerve Function. eNeuro 9:N/A (2022). PubMed: 35027445
- Kim YK et al. Transcriptome Profile in the Mouse Brain of Hepatic Encephalopathy and Alzheimer's Disease. Int J Mol Sci 24:N/A (2022). PubMed: 36614117
- Nitta A et al. Schizophrenia-Like Behavioral Impairments in Mice with Suppressed Expression of Piccolo in the Medial Prefrontal Cortex. J Pers Med 11:N/A (2021). PubMed: 34206873
- Sumiyoshi T Psychoneurobiology Research and Personalized Treatment of Schizophrenia. J Pers Med 11:N/A (2021). PubMed: 34945791
- Ackermann F et al. Critical role for Piccolo in synaptic vesicle retrieval. Elife 8:N/A (2019). PubMed: 31074746
Images
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Piccolo antibody (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.
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Western blot - Anti-Piccolo antibody (ab20664)All lanes : Anti-Piccolo antibody (ab20664) at 1 µg/ml
Lane 1 : Brain (Rat) Tissue Lysate
Lane 2 : Cerebellum (Rat) Tissue Lysate
Lane 3 : Kidney (Rat) Tissue Lysate (Negative control)
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 520 kDa
Observed band size: 460 kDa why is the actual band size different from the predicted?
Additional bands at: 200 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 4 minutesThe banding pattern shown in the image above is consistent with the literature which describes multiple bands >420 kDa as a result of proteolysis (PMID : 10707984).
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes using lysates heated to 85°C before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab20664 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
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Immunohistochemistry (PFA fixed) - Anti-Piccolo antibody (ab20664) This image is courtesy of Sophie Pezet, King's College London, United KingdomImmuofluorescent 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.
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Western blot - Anti-Piccolo antibody (ab20664) This image is courtesy of Randal Moldrich, CNRS UMR7637, ESPCI, FranceAll lanes : Anti-Piccolo antibody (ab20664) at 0.2 µg/ml
Lane 1 : Rat brain lysate at 40 µg
Lane 2 : Mouse brain lysate at 50 µg
Secondary
All lanes : 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 minutesPrimary 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.
