Microinjection of Proteins Protocols Protocol Links
Design, Synthesis, and Characterization of Caged Compounds Protocol When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected.
Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol Activation and inactivation of proteins using photoactivation of caged peptides or proteins offer insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group.
Microinjection of Protein Samples Protocol This protocol describes a method for microinjecting proteins into the nucleus or cytoplasm of adherent cells. Microinjection equipment can be obtained from a number of suppliers; this protocol has been used with the Narishige IM-200 air pressure regulator and the Leitz micromanipulator. Using this system, it is possible to microinject a constant volume within a 50% difference among cells.
Preparation and Loading of Protein Samples for Microinjection Protocol his protocol provides methods for the preparation of protein samples and for loading them into pulled microinjection pipettes. Stock solutions of proteins are thawed, diluted (if desired), centrifuged at high speed to remove aggregates, and kept on ice until loading. Loading into micropipettes can be done using either a "front-loading" or a "backfilling" procedure.
