Analysis of Protein Oligomerization by Electrophoresis.
A polypeptide chain can interact with other polypeptide chains and form stable and functional complexes called "oligomers." Frequently, biochemical analysis of these complexes is made difficult by their great size. Traditionally, size exclusion chromatography, immunoaffinity chromatography, or immunoprecipitation techniques have been used to isolate oligomers. Components of these oligomers are then further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by immunoblotting with specific antibodies. Although they are sensitive, these techniques are not easy to perform and reproduce. The use of Tris-acetate polyacrylamide gradient gel electrophoresis allows the simultaneous analysis of proteins in the mass range of 10-500 kDa. We have used this characteristic together with cross-linking reagents to analyze the oligomerization of endogenous proteins with a single electrophoretic gel. We demonstrate how the oligomerization of p53, the pyruvate kinase isoform M2, or the heat shock protein 27 can be studied with this system. We also show how this system is useful for studying the oligomerization of large proteins such as clathrin heavy chain or the tuberous sclerosis complex. Oligomerization analysis is dependent on the cross-linker used and its concentration. All of these features make this system a very helpful tool for the analysis of protein oligomerization.
Departament de Ciències Fisiològiques II, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Campus de Bellvitge, Barcelona, E-08907, Spain.
Monica
M
Cubillos-Rojas
Departament de Ciències Fisiològiques II, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Campus de Bellvitge, Barcelona, E-08907, Spain.
Taiane
T
Schneider
Departament de Ciències Fisiològiques II, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Campus de Bellvitge, Barcelona, E-08907, Spain.
Susana
S
Sánchez-Tena
Departament de Ciències Fisiològiques II, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Campus de Bellvitge, Barcelona, E-08907, Spain.
Ramon
R
Bartrons
Departament de Ciències Fisiològiques II, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Campus de Bellvitge, Barcelona, E-08907, Spain.
Francesc
F
Ventura
Departament de Ciències Fisiològiques II, IDIBELL, L'Hospitalet de Llobregat, Universitat de Barcelona, Campus de Bellvitge, Barcelona, E-08907, Spain. joseluisrosa@ub.edu.
Jose Luis
JL
Rosa
D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015151:Immunoblotting; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D011506:Proteins
Electrophoresis; Gradient gel; Oligomerization; PAGE; Protein cross-linking; Tris-acetate