[3dem] Rotary shadowing of coiled coil
guenter.resch at imba.oeaw.ac.at
Fri Feb 18 04:11:21 PST 2011
not exactly a 3DEM question, but maybe someone can give some feedback
on this nevertheless.
We are trying to visualise a dimeric protein by rotary shadowing; it
has a globular part of 2x 20 kDa and a 30-35 nm coiled-coil tail.
With the conditions we use so far (spraying onto mica from a 30%
glycerol, 100 mM ammonium acetate buffer; shadowing with 0.7-1.0 nm
Pt/C from an electron beam gun at 4-7 deg) we can nicely see the "head"
and sometimes also the tail. What surprises me is that in the very same
field of view, particles with the tail clearly visible can be found next
to others with no hint of it at all.
How can that be? Unfortunately, reliable visualisation of the tail
is essential for interpretation of this experiment. What conditions
would people experienced in rotary shadowing choose next for an
improved result? Change the angle? Shadow with W?
Thanks and best regards,
Dr. Guenter Resch, IMP-IMBA-GMI Electron Microscopy Facility
Email: guenter.resch at imba.oeaw.ac.at; Web: http://cores.imp.ac.at/em
Phone: +43 (1) 79044-4250; Fax/Voice Mail: +43 (1) 79044-224250
Post: Dr. Bohr-Gasse 3, 1030 Vienna, Austria, European Union
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