Microscale purification of proteins exhibiting anomalous electrophoretic migration: application to the analysis of GAP-43 phosphorylation.
Quite often, in vivo analysis of posttranslational protein modifications is complicated by the lack of specific antibodies or unsatisfactory immunoprecipitation efficiency. Here, we present an alternative method to immunoprecipitation that takes advantage of the anomalous electrophoretic behavior exhibited by GAP-43. This method can be applied to other proteins which show similar characteristics, such as myristoylated alanine-rich C kinase, NAP-22, and Neurogranin, among others. All these proteins display relative mobility values that depend on the concentration of polyacrylamide used in the resolving gel. Cell extracts or tissue homogenates are first separated by SDS-PAGE in 15% polyacrylamide gels, and the bands containing GAP-43 are identified, excised from the gel, and rerun on a second SDS-PAGE in 7.5% polyacrylamide/6 M urea gels. To quickly identify the position of GAP-43 in the first gel, a small amount of fluorescein-labeled recombinant GAP-43 was added to the initial extracts. The method, applied to the analysis of GAP-43 phosphorylation in rat hippocampal slices, can be typically completed in less than 4 h. The excellent yields of purification obtained contributed to a greater accuracy and increased reliability of the radioactivity measurements. It also allowed further processing of the samples, including the analysis of the different phosphorylation sites by proteolytic digestion and peptide mapping.
Tejero-Díez
P
P
Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, Madrid, 28049, Spain.
Rodríguez-Sánchez
P
P
Díez-Guerra
F J
FJ
D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019922:GAP-43 Protein / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008832:Microchemistry / Q000379:methods; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D051381:Rats
D019922:GAP-43 Protein; D003546:Cysteine Endopeptidases; C019913:clostripain