how to see low kd protein in vitro transcription/translation? - (Feb/27/2007 )

my protein is about 3 kd (only 2 methionine can label 35S)

and i use pcDNA3.1 plasmid insert my protein's nucleotide sequence to do in vitrotranscription translation

and i use 15% and 20% SDS-PAGE to run western but i can't see my protein

can some one help me?

change my condition or use different gel

thanks a lot
by len

hmmm

for Western, it may be about concentration

are you sure you have a lot of transcription?

the other question, what sort of membrane do you use? what is the pore size? it is best to use 0.2um PVDF for tiny proteins, and in my experience wet transfer is a bit better than semi-dry (it's easy to lose a tiny protein even with a very quick transfer)

would you mind posting your protocol?

For low-molecular proteins use only Tricine PAGE (16% or 10-20% gradient better). on SDS-PAGE you can't detect your 3 kDa protein because of comigration of SDS micelles with small polypeptides If you want I can send your protocol.

i use promega TNT@quick coupled reticulolyte lysate system

50μl total reaction

40μl reticulolysate(include all the reagent) + 1μl 35S + 1μl plasmid(about 1 μg plasmid) + 8 nuclease free water

i have a positive control and i can see it on my film so i think the transcription efficiency is ok

thanks for your reply

by len