Dear all,<br><br>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.<br><br>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.
<br><br>- Deposit thicker carbon films (Mark Rosenberg, Ricardo Bernal). Didn't help us.<br>- 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.
<br>- Use 1000 mesh grids or holeyfilms so small areas stay intact (Rachel Scherer).<br>- Work with a different detergent. DDM and digitonin are good (Bettina Boettcher, John Rubinstein).<br>- Put a plastic film between the copper grid and the carbon film (Bettina Boettcher, Ed Gogol).
<br>- 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.
<br>- Be very diligent when you prepare the carbon films (Reinhard Rachel, Guy Schoehn).<br>- Make carbon film hydrophilic by glow-discharging grids (Ching-Ju Tsai).<br>- Dilute protein solution (and thus detergent) right before applying to grid. Might work if protein doesn't aggregate immediately (Matt
L.Walker).<br>- Good luck (all who responded). Thanks.<br><br>A special Methods issue on EM is forthcoming that will contain a review on EM of membrane proteins by John Rubinstein.<br><br><br>Andreas<br><br>