RNA Meditations

Promega Imagine a world without 35S methionine. It's easy with Promega's TranscendTM Non-Radioactive Translation Detection Systems. Utilizing Promega's novel biotinylated lysine tRNA, these systems can be used to detect in vitro translation products after blotting with a variety of detectors, including Promega's own TranscenTM chemiluminescent substrates. No more wipe tests, no more radioactive waste disposal headaches. And results can be obtained in half the time. One microliter of TNR transl

Nov 24, 1997
The Scientist Staff

Promega Imagine a world without 35S methionine. It's easy with Promega's TranscendTM Non-Radioactive Translation Detection Systems. Utilizing Promega's novel biotinylated lysine tRNA, these systems can be used to detect in vitro translation products after blotting with a variety of detectors, including Promega's own TranscenTM chemiluminescent substrates. No more wipe tests, no more radioactive waste disposal headaches. And results can be obtained in half the time.


One microliter of TNR translation reaction was spearated on a SDS-PAGE gel, blotted onto a PVDF membrane. The biotinylated proteins were detected with Streptavid-HRP and TranscendTM Chemiluminescent Substrates. (Lane 1 beta-galactosidase, Lanes 2 CAT and Lane 3 luciferase)
Promega has developed the TranscendTM lysine tRNA, which has a biotin moiety linked to the e-amino group of a lysine molecule for use in any in vitro translation system. The biotin is linked to the lysine via a spacer arm to facilitate detection with avidin/strepavidin detectors and can be used either as a detector or to capture the translated protein with streptavidin resins or membranes. After SDS polyacrylamide gel electrophoresis and blotting, biotin containing proteins can be detected by streptavidin-alkaline phosphatase or streptavidin-horseradish peroxidase followed either by colorimetric or chemiluminescent detection. Advantages over conventional detection systems include the stability of the biotin tag (up to twelve months, either as a tRNA molecule or incorporated into protein), the choice of detection methods, and the speed of detection, which takes only a few hours to complete. Finally, as lysine is more prevalent in proteins than the commonly used methionine (lysine represents 6.6% of a protein's amino acid content while methionine only represents 1.7%), this system will be useful for studies of proteins with low methionine content or in protein truncation tests.

Promega has looked at the effect of biotin on enzyme and expression levels of three proteins in a reticulocyte lysate system. Two of the three (luciferase and CAT) showed no or minimal effects of biotin, while beta-galactosidase activity and expression were reduced (53% of enzymatic activity in the worst case and 70% of synthesis relative to a control reaction). Estimates of incorporation levels indicated that 25-33% of lysine residues are biotinylated in translation products that contain 4-6 lysine residues.

The kit comes in several forms: the TranscendTM Colorimetric Translation Detection System, with sufficient reagents to label 30 translation reactions and to perform colorimetric detection of biotinylated proteins on six blots; the TranscendTM Chemiluminescent Translation Detection System, with sufficient reagents to label 30 translation reactions and perform chemiluminescent detection of biotinylated proteins on six blots; or the TranscendTM tRNA alone.

For more information, contact Promega at 800-356-9526 or visit their web site at www.promega.com.