Concentrating proteins for N-terminal sequencing - (Jul/21/2006 )

Does anybody have experience in N-terminal sequencing? I am trying to isolate a protein from the supernatant of a bacterial culture and my proteins are separating very well on SDS-gel but they are very faint! Do you know any efficient way of concentrating supernatant proteins!?

I have also read that it is not reccomended to make a double well on SDS to be able to load more proteins since the band you wish to send for N-terminal sequencing shouldn't be wider than 5mm which is the width of the cartridge for sequencing. True or false?

Any help is welcome, thanks!

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i know 2 methods for protein concentration...
Centricon columns or TCA/Acetone....
Second one is cheaper and maybe faster to set up but don't know if it's ok for your exp.

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The rule of thumb that I told my clients when I ran a core facility was "If you can see it on a gel by Coomassie staining, you have enough to sequence from". This is true if you transfer to membrane, too: if you can see it, you should be able to sequence it, unless the protein is N-terminally blocked.

As for the question of double-width lanes, the best thing is to check with whomever is going to do the sequencing. They can tell you what their preferred format is.

Seeing as how your proteins are going to be totally denatured anyway, Fred_33's idea to acetone ppte is good.

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