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Reagents & Solutions 800-356-9526 608-274-4330 enotes@promega.com
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FAQspeakHow can I increase the protein yield from my TNT® Coupled Reticulocyte Lysate System reaction?There are many factors that can affect protein yield in a TNT® coupled transcription/translation reaction. Because protein yield is dependent upon the nature of the DNA template, some factors may have a more pronounced effect than others for a particular template. First, the DNA template should be checked for the presence of additional AUG translation initiation codons encoded between the promoter and the initial start codon. Additional upstream start codons can cause a frameshift or a fusion protein, resulting in a protein of an unexpected size. Ideally the template would also include a poly(A) tail of at least 30 bases, since a poly(A) tail has been shown to increase protein yield (1). The 5´ untranslated region should be devoid of any strong secondary structures, which can cause the ribosome to stall and protein yield to decrease. The titration of potassium and/or magnesium into a TNT® reaction can cause a noticeable increase in yield. Optimal potassium levels for many templates is in the range of 100–135mM, while the optimal range for magnesium is 2.0–2.5mM. Increasing potassium concentrations in increments of 10–20mM and magnesium concentrations in increments of 0.5mM in a standard TNT® reaction is a good guideline to determine the optimal salt concentrations for a particular template. In general, PCR product templates require higher potassium and magnesium concentrations than circular templates. Finally, for proteins larger than ~100kDa, yield of full-length protein can often be increased by decreasing the reaction temperature from 30°C to 25°C. Reference
*Products may be covered by pending or issued patents. Please visit our patent and trademark web page for more information.TNT is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office. |
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