Rabbit Reticulocyte Lysate System Technical Manual, TM232

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X. Troubleshooting (continued) Symptoms Possible Causes Comments Low translation Calcium is present Avoid adding calcium to the efficiency of the in the translation reaction translation reaction. Calcium may sample mRNA reactivate the micrococcal nucle- (continued) ase used to destroy endogenous mRNA in the lysate and result in degradation of the mRNA template. Ethanol is present in the Residual ethanol should be translation reaction removed from mRNA preparations and labeled amino acids before they are added to the translation reaction. Unexpected bands are Denaturing temperature Denature sample at a lower present at higher is too high temperature (e.g., 60–80°C). molecular weights Unexpected bands are Proteolysis of translation Add a protease inhibitor, such present on the gel product as α-macroglobulin, leupeptin or chymostatin. More than one peptide Leaky scanning for translation is translated from the initiation can result in translation RNA template initiating at internal downstream methionines. 35S-labeled amino acid Older 35S may dissociate from the is beyond its expiration amino acid and label other proteins date in the lysate. Use fresh 35S. The [35S]methionine There are reports of a 42kDa band used is not of with some grades of translational grade [35S]methionine (3). We recommend Redivue L-[ 35S]methionine (Amersham Cat.# AG1094) to avoid this 42kDa band. Globin may appear Globin may show on a stained gel on the autoradiogram and occasionally as a faint image or stained gel on the autoradiogram. It appears as a broad band migrating at 10–15kDa. Aminoacyl tRNAs may Add RNase A to the lysate produce background reaction (after completion) to a bands (~25kDa) final concentration of 0.2mg/ml. Incubate for 5 minutes at 30°C. Oxidized β-mercapto- Use a loading buffer that contains ethanol is present or not 2% SDS and 100mM DTT. enough SDS loading buffer Low protein yield Mg 2+ concentration is Titrate the amount of Mg 2+ used not optimal in the reaction (22). K + concentration is Titrate the amount of K + used in not optimal the reaction (22). Amount of RNA used is Titrate the amount of RNA added not optimal (22). Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# <strong>TM232</strong> Printed in USA. Page 16 Revised 03/00
X. Troubleshooting (continued) Symptoms Possible Causes Comments Low protein yield Incubation of the reaction Incubate the translation reaction (continued) at 37°C can decrease at 30°C. protein synthesis The addition of With the addition of spermidines polyamines, such as and diamines, the optimal Mg2+ spermidine and certain concentration will change. diamines, has been Therefore, these components shown to stimulate must be co-optimized. translation (10) Reaction time may need Increase reaction time to to be optimized 90–120 minutes. There is smearing Gel not clean Gel must be washed before on the gel placing onto film. Once gel electrophoresis is complete, soak the gel in either a standard Coomassie® destaining solution (50% methanol, 7.5% glacial acetic acid) or in water for 15–30 minutes prior to drying. Too much protein loaded Check the amount of samples on the gel loaded on the gel and the amount of loading buffer. Too much protein loaded on the gel can cause smearing. Acrylamide Acrylamide concentration can be concentration too low to increased to 12%. resolve proteins Ethanol present in the Ethanol present in the RNA RNA sample sample can cause smearing on the gel. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · Telephone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# <strong>TM232</strong> Revised 3/00 Page 17
Page 1 and 2: Rabbit Reticulocyte Lysate System T
Page 3 and 4: C. Applications Potential applicati
Page 5 and 6: Transcend Systems, refer to the Tra
Page 7 and 8: free from DNA contamination. Promeg
Page 9 and 10: Thus, reaction parameters may need
Page 11 and 12: 5. Place the polyacrylamide gel in
Page 13 and 14: Ideally, the coincidence circuit of
Page 15: X. Troubleshooting Symptoms Possibl
Page 19 and 20: B. Related Products Translation Sys
Page 21 and 22: RNA Production Systems Product Size
Page 23 and 24: 22. Beckler, G.S. et al. (1995) In:

Rabbit Reticulocyte Lysate System Technical Manual, TM232

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