In vitro translation
T McIntosh - Biology
tmcintos at flash.LakeheadU.Ca
Wed Jul 6 14:36:18 EST 1994
I am in desparate need of help with getting a wheat germ translation system working. I am the 3rd person in this lab to work on it and none of us have been able to get it running.
I have used several methods including the Boerhinger Mannheim translation kit and Erickson and Blobel's protocol with and without modifications to precipitation and washing procedures.
Boerhinger's protocol calls for discharging tRNas with sodium hydroxide, precipitating with 2% casein?25% TCA and washing unincorporated aas off of GFC filter papers with sodium pyrophosphate in TCA
under vacuum filtration. Erickson and Blobel use hot TCA precipitation. I've also used cold TCA precipitation, and several types of filter papers.
As far as the actual translation system goes, I've tried changing Wheat germ, mRNA,K, Mg and ATP concentrations to get better incorporation rates. Using either BMV or TMV mRNa I have never
gotten more than double the incorporation over the negative control. I have also tried translating a di-ubiquitin mRNa that I transcribed, but with not much better luck.Incorporation goes up at about
20 minutes but by thirty minutes it goes back down and continues to dive. Adding calf thymus tRNA, protease inhibitors and Placental RNase Inhibitors don't help much either. I've even added a wheat
germ ribosomal fraction to the system to enrich the concentration of initiation factors. Nothing seems to work.
On SDS gels I can see that proteins are being translated above control levels, but it's the kinetics of translation that I'm interested in, so it's incorporation rates that I need. I've used
both 14C-leu and 35S-met, and settled on the latter due to overall higher counts.
Does anybody have an idiot proof protocol for translation using the wheat germ system that is tried and true? I feel like I must be missing something basic for it not to be working and there is
nobody on campus who has any experience with translation systems.
If anybody can help please e-mail me. My address is tmmcinto at cs_acad_lan.lakeheadu.ca
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