Reversible fixation for the study of morphology and macromolecular composition of fragile biological structures.
Many subcellular structures are assemblies of subunits, which are in dynamic equilibrium with free subunits in solution. Their dissociation upon dilution, resulting from cell lysis, can be prevented by adequate fixation. If the latter is reversible, the constituting proteins of such subcellular structures can be analysed electrophoretically. Polyheads of bacteriophage T4 are in dynamic equilibrium with their subunits. They fit very well as a probe to measure the efficiency of crosslinking by the arrest of dissociation upon dilution and upon treatment with hot SDS. Formaldehyde (5%, 20 min, 20 degrees C) leads to a stabilisation comparable to fixation with glutaraldehyde (1%, 30 min). The fixation is shown to be reversible up to 86% by acid and borohydride treatment, but is stable towards heat and SDS-mercaptoethanol. Bands of the reversed proteins are neat and not found in detectably different positions in comparison to controls, when checked by SDS gel electrophoresis.
D004591:Electrophoresis, Polyacrylamide Gel; D005404:Fixatives; D005557:Formaldehyde; D005976:Glutaral; D008828:Microbiological Techniques; D013604:T-Phages / Q000648:ultrastructure*
D005404:Fixatives; D005557:Formaldehyde; D005976:Glutaral