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Synthetic peptide conjugated to KLH derived from within residues 600 - 700 of Rat Piccolo.
(Peptide available as ab23400.)
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 concentration. 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 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.|
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.
This image is courtesy of Randal Moldrich, CNRS UMR7637, ESPCI, France
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.