Western Blotting Of Camkii Β And T 287

847 WordsOct 20, 20154 Pages
Western blotting of CaMKIIβ and T-287 phosphorylated CaMKIIβ Western blotting was performed as described previously (Chapter 3). In short, Proteins were extracted from OLs using RIPA buffer with protease and phosphatase inhibitors. The concentration of extracted proteins was assessed using the BCA assay. Target proteins were probed with primary antibodies for CaMKIIβ (1:1000, Life Technologies) and phosphor-CaMKIIβ (T287) (1:1000, Abcam) at 4°C overnight. Membranes were incubated with appropriate IRDye secondary antibodies (1:3000, Li-COR, Lincoln, NE) for 1 h at room temperature, and imaged using an Odyssey Imager (Li-COR). Protein bands were quantified using Li-COR image studio software. RT-PCR analysis of CaMKII transcription RNAs were extracted from OLs or cerebral cortex of P2 ICR mice using the miRNAeasy Mini Kit (QIAGEN Inc. Valencia, CA). Concentration of RNAs from each sample was determined using NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Equal amount of RNAs were then used as template to transcribe cDNA using a High capacity cDNA reverse transcription kit (Life Technologies). Gene-specific primer pairs were designed using NCBI Gene database and primer design web tool (http://www.ncbi.nlm.nih.gov). The web tool generates a series of primer sets that fit the parameters defined by users. These parameters include size of the final product and the primers, whether primers span the junctions between exons, GC contents of the primer etc. For each isoform

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