I am trying to show in vitro that my protein can bind tRNA (as a control). The protein is of human origin and I think it would be best to use human tRNAs. Currently it is hard to order human tRNAs and I have not been able to find many protocols on how to either isolate human tRNAs (preferably charged) or how I would go about running an EMSA when I acquire it (i.e how do I make sure the tRNA is folded correctly).
The best thing is to isolate it out of real tissue. Owing to the close similiarity between cows and humans, your best bet is to find a good old time paper that isolates tRNA out of beef liver. Grind up tissue, extract with water saturated phenol, precipitate with ion ethanol, resuspend and run over ion exchange column (eg monQ). This will be a lot more accurate than using transcripts, which lack modifications
You can synthesize RNA in vitro (transcription). It will be deprived of nucleotide modifications. Maybe it will be suitable for your EMSAs.
Thank you. I found a paper that uses a yeast tRNA (sold by Sigma) for the control experiments but I think I will also in vitro transcribe as well and then charge the tRNA. Thank you again.
The best thing is to isolate it out of real tissue. Owing to the close similiarity between cows and humans, your best bet is to find a good old time paper that isolates tRNA out of beef liver. Grind up tissue, extract with water saturated phenol, precipitate with ion ethanol, resuspend and run over ion exchange column (eg monQ). This will be a lot more accurate than using transcripts, which lack modifications
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA