Evaluation of epitope tags for protein detection after in vivo CNS gene transfer.
Functional characterization of disease-related proteins, their splice variants and dominant negative mutants in the context of complex CNS tissues such as brain and retina is frequently assessed by in vivo gene transfer. For correct interpretation of results it is imperative that the protein under investigation is unambiguously detected in the transduced cell types and can be distinguished from any endogenously expressed physiological variants. Therefore the first systematic evaluation of epitope tags used to trace ectopically expressed proteins in the central nervous system is presented here. Substantial differences in the performances of various epitope tag-antibody combinations with respect to sensitivity, specificity and influence of the epitope tag on the fusion protein are elucidated. Epitope tags already established for protein detection in vitro and to some extent in vivo (c-Myc, HA and FLAG tags) were immunohistochemically detected with high sensitivity. However, detection of these tags revealed problems with background staining and we also document structural and functional influence of the tags on the fusion protein. In order to prevent such unwanted side-effects, epitope tags which have not yet been used for in vivo applications (IRS, EE and AU1 tags) were characterized in brain, retina and cultured neurons. While use of the IRS and EE tags was hindered by low sensitivity or specificity, optimal results were obtained with the AU1 epitope, which may develop into a standard tool for detection of ectopic protein expression in the central nervous system.
Shevtsova
Z
Z
Department of Neurology; University of Göttingen, Medical School, Waldweg 33, 37073 Göttingen, Germany.
Malik
J M I
JM
Michel
U
U
Schöll
U
U
Bähr
M
M
Kügler
S
S
D000667:Ampicillin / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D064026:Calbindins; D002490:Central Nervous System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics*; D005260:Female; D018390:Gene Targeting; D005822:Genetic Vectors / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism
D064026:Calbindins; D000939:Epitopes; C107055:N-(6,7-difluoroquinolonyl)ampicillin; D009842:Oligopeptides; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D049452:Green Fluorescent Proteins; D000667:Ampicillin; C085706:FLAG peptide