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TNT coupled in vitro translation


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#1 saad

saad

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Posted 01 June 2009 - 12:56 AM

Hi everyone

I need a help, actually I've done in vitro translation by using TNT in vitro couple transcription/translation with plasmid that is prepared inhome by miniprep.
- I use 35S Met to label proteins (prey and bait)
- incubation at 30C for 2 hr
- incubate the flag-protein with anti-flag beads first for 2 hr then incubate with bait protein o/n at 4C, washing 6x . add 20 ul 4X SDS heat 10 min at 70 C then load 15-20 ul on SDS-PAGE. I expose the gel using Phosphor Screen then scan with Typhoon.
I've problem with unexpected bands and some strange results and I hope to optimise this experirment.
for instance how much volume of beads"difficult to adiust the volume of beads", I use usually 25-30 ul then start equilibrate &washing
how much volume should I load on the gel?
any suggestion welcome and I appreciate every comment.

Thanks

Edited by saad, 01 June 2009 - 12:59 AM.





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