Thank you!
Maria Lorenzi (mlorenzi@ascu.unian.it)
Sat, 7 Nov 1998 18:01:35 +0100
Hi, I'm Maria.
First of all I thank everybody for the answers that you gave to me.
My Sequencer is a ABI 491 and unsfortunately I don't have in my laboratory a
mass spectrometry.
My protein hasn't been purified by SDS-PAGE and, before loading the sample
in the Sequencer, I denatured the protein with SDS and reduced it with DTT;
then I alkylated it with acrylamide.
In this way, during the sequence, I saw the modified cysteines that elute
immediately after E but, in the correspondent cycles, I didn't see the two
peaks that I mentioned to you.
I saw them in the cycle of supposed tryptofan and they have a different
ritention time from the modified cysteines.
I cannot see the tryptofan during the sequencing of the protein but I see it
in the standard cycle; I can reduce the conversion flask dry-down time but I
don't think that it can improve the situation.
I want to add DTT in the solvents as Ricardo Bastos Cunha suggested me.
Using the formula that Dan Brune gave to me about the molar extinction
coefficent of the protein, it seems that my protein has one tryptofan.
I have an analyzer of amino acids in my laboratory but it is very old and it
take too much time to use it.
I have other questions for you.
I would like to know what kind of modifications a protein can have and how
much they can influence the MW of it.
In addition, I desire to know where I can find some informations about the
codon usage in the fungus; I'm studing a toxin from the fungus Phytophthora
cactorum and I' m planning to clone the gene of the protein.
Thanks a lot for your help
Ciao
Maria