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.
The 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.
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.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Piccolo antibody (ab20664)This image is courtesy of an abreview submitted by Karine Thibault, King's College London, United Kingdom
IHC-FoFr image of Piccolo staining on rat brain cortex tissue sections. The tissues were fixed (animals perfused fixed) with 4% PFA and later postfixed overnight in the same fixative. They were cryoprotected in 30% sucrose and cut using a cryostat. The staining was performed using free floating technique.
Immunohistochemistry (PFA fixed) - Anti-Piccolo antibody (ab20664)This image is courtesy of Sophie Pezet, King's College London, United Kingdom
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.
Western blot - Anti-Piccolo antibody (ab20664)This image is courtesy of Randal Moldrich, CNRS UMR7637, ESPCI, France
All 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 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.