Application of proteomic tools to detect the nonspecificity of a polyclonal antibody against lipoprotein lipase.
Specific antibodies are essential tools for studying proteins. P66 is a chicken polyclonal antibody raised against bovine lipoprotein lipase (LPL) that has been used in earlier studies. Here, we developed a two-dimensional (2D) Western blot with reducing gels, using commercial bovine LPL (53 kDa) as a standard and P66 for detection. Our results revealed incomplete purification of commercial LPL and nonspecificity of P66, both undetectable in one-dimensional analysis. Antithrombin III (ATIII) was identified as both a major contaminant in commercial LPL and a cross-reacting protein with P66. Although LPL purification methods were presumably designed to eliminate ATIII, here we demonstrate that some procedures fell short of this objective and thus led to the production of a nonspecific antibody. Our results define 2D electrophoresis/Western blot and mass spectrometric protein identification as the most reliable procedure for validating LPL purity and the specificity of antibodies against this enzyme.
Casanovas
Albert
A
Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, E-08028 Barcelona, Spain.
Carrascal
Montserrat
M
Abián
Joaquín
J
López-Tejero
M Dolores
MD
Llobera
Miquel
M
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D008071:Lipoprotein Lipase / Q000276:immunology*; D040901:Proteomics
D000906:Antibodies; D008071:Lipoprotein Lipase