In article <3626E881.5067973F at student.mahidol.ac.th>,
g3937506 at STUDENT1.MAHIDOL.AC.TH (Jongrak Kittiworakarn) wrote:
> Is it possible to do as follows:
> First, separate the peptide from other peptides/proteins by
> electrophoresis. Then, transfer it to nitrocellulose membrane, and cut
> the band of interest. Dissolve the membrane in acetone and separate the
> peptide by precipitation.
I am going to assume that you will give this protein to someone else who
will actually do the sequencing. It is therefore best that you ask them
how they wanted the protein given to them. Many labs would prefer to take
the proteins off the membranes themselves. With that said, here is the
procedure that I use for MS sequencing:
1) transfer protein to PVDF (sequencing grade: It has smaller pores and
more surface area) in Tobin buffer + DTT if free cysteines exist.
2) W/o drying membrane wash with LARGE amts of pure water to remove
SDS(very nasty to MS), glycine and tris.
3) stain in 0.1% ponceau red in 3 % acetic acid for 5 -10 min. Then wash
with ure water to remove excess stain. To much acetic acid can dehdrate
some aa's making spectra harder to interpret. Also, this stain is not
sensitive and works like crap on glycosylated proteins BUT it is easy to
remove. CB is not easy to remove and can make MS spectra more
complicated.
4) Cut out band of interest and remove stain by breifly dipping the
membrane in 200uM NaOH then immediately follow with lots of
water.(prolonged exposure to high pH will cause your protein to wash off
the membrane)
5)place the membrane in 100mM ammonium acetate (pH8) overnight and chop
the membrane up. (hydrophobic and large proteins might not come off, if
this is a problem then increase the pH)
6) wsh the mebrane for 4-6 hrs (twice) with 100 uL of above buffer. Then
reduce it to a suitable olume in lyophilizer and load onto RP-capillary
column which is eluted straight into the MAss Spec.
Good luck,
Peter
--
"don't you eat that yellow snow
Watch out where the huskies go"
FZ
