finding GFP's mass

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Apr 13 14:54:03 EST 2001


"Emir Khatipov" <ekhatipo at NOSPAMmidway.uchicago.edu> wrote:
:
:I think your question was mainly about whether there are other "wetlab"
:methods  of determination of molecular weight of a protein. In addition to
:SDS-PAGE you used, there is also 

:native electrophoresis, 

That's what my boss is also saying, but this is very wrong.
Native EF has everything to do with charge and almost 
nothing with size.

:gel filtration, ultracentrifugation, mass spectrometry, chemical methods, 
:like Kjeldal analysis, and others.

Whta's Kjeldal analysis? Never heard of it. (Should have?)

        - Dima

:"Frederic Langlois" <ye.midget.pirates.deleted.my.account at the.seven.seas>
:wrote in message news:eppz6.12265$up4.324633 at carnaval.risq.qc.ca...
:> Hello.. I'm trying to figure out the different ways to calculate a
:protein's
:> mass for an assignment. In this particular case I want to know the mass of
:> the green fluorescent protein (GFP).
:>
:> 1) The first method I have used is the SDS-PAGE electrophoresis with the
:low
:> range standard markers. I made a standard curve of the standards Rf value
:(Y
:> axis) and the molecular weight (X axis). I extrapolated the GFP's mass
:using
:> it's Rf value.
:>
:> 2) Then I searched many databanks and found GFP's amino acid sequence. I
:> used this equation to find GFP's mass:
:>
:> mass = [(n1*molecular
:>
:weight1)+(n2*mol.weight2)+...(n20*mol.weight20)]/[(n1+n2+...n20-1)*33g/mol]
:>
:> where 1=glycin; 2 to 20= all the other amino acids
:>       n=the sum of the amino acids of each kind
:>       33g/mol=a water molecule's molecular weight (approximately)
:>
:> I substrated the water molecules that are "lost" when the peptidic bonds
:are
:> made.
:>
:> I converted the molecular weight in Dalton units...
:>
:> 3) what are the other methods I can use to find the mass of this protein?
:>
:> Thanks in advance,
:>
:> Frederic Langlois
:>
:>
:>
:
:




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