Some specimens, such as membrane proteins, have to be solubilized in a detergent and these are always difficult to work with although some successful results have been obtained. Some specimens which have met all the above criteria simply do not work out. An occasional specimen will not attach to the carbon film. Double-stranded DNA, for example, does not attach reliably even at high concentrations. Polylysine pre-treatment will also help some other types of specimens.
All proteins denature to some extent at the air-water interface of the drop on the grid.
Usually, washing and wicking removes this denatured film. However, some proteins continue to denature at the interface until there is nothing left on the grid. Sometimes washing with low concentrations of organic solvents such as ethanol or acetonitrile will help. Some specimens which fall apart on the carbon film benefit from a brief fixation with glutaraldehyde just prior to injection. Occasionally specimens will be all right on some parts of the grid but not in other areas.