Transcend biotin lysyl trna

In vitro generated RNA transcripts above were used templates for in vitro translation assays using a Rabbit Reticulocyte Lysate kit (Promega) at the indicated concentrations. Reactions were performed at 1/5^th volume with the inclusion of Transcend Biotin-Lysyl-tRNA (Promega) to allow antibody independent detection of all translated products. Samples were assayed by western blot (as above) with the exception that membranes were blocked with 5% BSA/TBS for 1 hour, incubated with IRDye 800CW Streptavidin (LICOR) for 1 hour, washed 3 times and visualised using the LICOR Odyssey Fc imaging system.

A MYLK3 ORF clone generated by GenScript (Clone ID: OMu23765) in the pcDNA3.1(+) vector was used as the backbone for subsequent 5′UTR cloning. 5′ UTR of B6N and B6J MYLK3 was cloned immediately upstream of the canonical ATG and immediately downstream of the T7 promoter by Gibson Assembly (89 (link)) and confirmed by Sanger sequencing. 1 μg of plasmid was added to 40 μl of TnT Quick Master Mix (Promega) with 1 μl of methionine and 1 μl of Transcend Biotin-Lysyl-tRNA and incubated at 30°C for 1 h. A negative control reaction was set up containing no plasmid DNA, and a positive control containing a luciferase expression vector. The products of each reaction were separated by SDS–PAGE and then transferred onto a nitrocellulose membrane. The membrane was blocked in 5% BSA and incubated with MYLK3 and detected by Western blot protocol or with streptavidin–HRP conjugate and incubated for 2 h, followed by detection with substrate as per the manufacturer’s instructions (Promega). Membranes were visualised using a Licor Odyssey Fc and ImageStudio software.