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N-terminal sequencing


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#1 chn09

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Posted 26 November 2009 - 02:38 AM

Dear all,
I have to perform an N-terminal sequencing of the protein expressed in E. ColiBL21. But the protein is expressed as inclusion bodies. Even though i puriifed the protein suing 8m urea under denaturing condition the yeild is very low on an SDS-gel. The band of the purified protein is faintly visible. Is it enough for N-terminal sequencing? Now i have 2 options

1. Cut out the band after blotting it on PVDF membrane and send it for N-terminal sequencing

2. Run an SDS-PAGE of the purified inclusion bodies , then blot it onto PVDF, cut out the band corresponding to the overexpressed protein and the send it for N-terminal sequencing .
Which of these should i do? Pls help

#2 Prep!

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Posted 26 November 2009 - 02:57 AM

if your protein is more than 98% pure u can directly blot it on the membrane and send for sequencing...
generally around 100 pmol is enough for sequescing 10 residues!!
hope it helps!!
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#3 mdfenko

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Posted 26 November 2009 - 09:46 PM

Dear all,
I have to perform an N-terminal sequencing of the protein expressed in E. ColiBL21. But the protein is expressed as inclusion bodies. Even though i puriifed the protein suing 8m urea under denaturing condition the yeild is very low on an SDS-gel. The band of the purified protein is faintly visible. Is it enough for N-terminal sequencing? Now i have 2 options

1. Cut out the band after blotting it on PVDF membrane and send it for N-terminal sequencing

2. Run an SDS-PAGE of the purified inclusion bodies , then blot it onto PVDF, cut out the band corresponding to the overexpressed protein and the send it for N-terminal sequencing .
Which of these should i do? Pls help

is the band visible with coomassie? if so, then it is probably enough to sequence, so you can do 1.

if not, then run the inclusion bodies (you could do this either way).
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