No compartment for proteins:an approach for isolating differentially located Intermediate filaments

44 FEBS Congress, Krakow, Poland, 06 July 2019, pp.424

Desmin is a muscle-specific intermediate filament (IF) protein

located in cytoplasm. Lamin B on the other hand is a type V IF

protein located under the nuclear envelope and is farnesylated to

re-enforce the membrane connection. The isolation of IF proteins

is challenging since they are elongated and highly polymerized

structures. Isolation procedure for IFs frequently requires denaturing

conditions. However, our aim was to isolate desmin and

lamin B while preserving native protein structure for downstream

assays. Zebrafish (Danio rerio) skeletal muscle tissue was used for

protein isolation. The tissue was first mechanically disrupted via

mortar and pestle followed by sonication. An isolation buffer

containing both ionic and non-ionic detergents, and reducing and

chelating agents, was used for chemical disruption. A dialysis

step was added to procedure to remove reducing agent since the

lysate was intended to use for co-immunoprecipitation. The problem

regarding our co-immunoprecipitation assay was risen from

the fact that the targeted proteins are located in the biochemically

and biophysically diversified compartments generated by

nuclear envelope structure. Combining the compartmentalization

“problem” with the entangled organization of IFs, finding the

proper isolation method was a real obstacle. By using this

method, isolation of two differentially located IF proteins in a

non-denaturing condition was successfully achieved. This study

had been funded by the Scientific and Technological Research

Council of Turkey, Project number 214S174 to P. Dincer.