[3DEM] [summary] grids and detergent

[Andreas Forster]

Dear all,

two days ago I asked the ccp4 and 3DEM lists how one could prevent carbon
films from being destroyed by detergent.  At least that's what I meant to
ask.  Thanks for the many responses I got in no time (summarized below).
Thanks to all who responded and apologies to those I don't mention.  All
suggestions have been relayed to our EM wizard.

Most asked what I meant by 'eats the carbon grid' and guessed correctly that
the carbon film explodes into little pieces when sample is applied between
mica and film.  The film suffers the same sad fate when it is adhered to the
copper grid first.

- Deposit thicker carbon films (Mark Rosenberg, Ricardo Bernal).  Didn't
help us.
- Use glue (e.g., strip from clear tape with chloroform) to hold the carbon
film to the copper mesh (Richard Easingwood, Guy Schoehn).  Be careful not
to fill the holes in the grid.
- Use 1000 mesh grids or holeyfilms so small areas stay intact (Rachel
Scherer).
- Work with a different detergent.  DDM and digitonin are good (Bettina
Boettcher, John Rubinstein).
- Put a plastic film between the copper grid and the carbon film (Bettina
Boettcher, Ed Gogol).
- Instead of applying the sample between mica and carbon film and then
adhering the carbon film to the grid, adhere the carbon to the grid first,
dry well and apply sample (Daphna Frenkiel-Krispin).  Didn't help us.
- Be very diligent when you prepare the carbon films (Reinhard Rachel, Guy
Schoehn).
- Make carbon film hydrophilic by glow-discharging grids (Ching-Ju Tsai).
- Dilute protein solution (and thus detergent) right before applying to
grid.  Might work if protein doesn't aggregate immediately (Matt L.Walker).
- Good luck (all who responded).  Thanks.

A special Methods issue on EM is forthcoming that will contain a review on
EM of membrane proteins by John Rubinstein.


Andreas
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