finding GFP's mass

Dr. Artem Evdokimov eudokima at mail.ncifcrf.gov
Fri Apr 13 16:06:59 EST 2001


> :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.

Precisely. Native phoresis is great when you are concerned about
homogeneity of the protein sample (something that looks OK on SDS PAGE
often looks horrible on native gel) for the purposes of protein
crystallization. It sucks as m.w. tool.

The thing to do for decent 'rough' m.w. in solution is static light
scattering (12-18 angles) combined with refraction index measurements -
this method gives ~1KDa accuracy in many cases. (I don't believe
DynaPro's dynamic light scattering machines - I've seen enough
artifacts).

Best thing by far is ESI-MS or MALDI-TOF but that takes expensive
equipment. MS can give you under 1 Da accuracy for a reasonable-sized
protein.

Ultracentrifugation is quite accurate indeed but it takes time.

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

If you were a chemist you probably would have heard of this method -
Kjeldahl's total nitrogen analysis. It is not trivial to figure out
protein m.w. from that, unless you have other helpful data. Normally
this is done to confirm the general sequence, not to calculate molecular
weight.

A.

-- 
|Dr. Artem Evdokimov   Protein Engineering |
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