Comprehensive polypeptide analysis of microdissected rat brain areas: combining 2-dimensional gel electrophoresis with 2-dimensional HPLC and immunoanalysis and sequencing procedures.
An improved methodology has been developed which allows resolution, identification, and quantitation of hundreds of proteins and neuropeptides from a single rat brain nucleus (5 mg wet wt.). After metabolic labelling, proteins (greater than about 15 kDa) are separated from peptides (less than about 10 kDa) by sonicating the tissue in an acidic peptide extraction buffer; after centrifugation, proteins are in the pellet, peptides in the supernatant. To quantitate peptide synthesis, peptides are resolved to purity by reverse-phase HPLC followed by ion exchange HPLC. Proteins are resolved with a two-dimensional (2-D) gel protocol optimized for neural tissue. To identify specific proteins by immunoanalysis, proteins are transferred to polyvinyl difluoride (Immobilon) and immunostained in the presence of Tween blocking buffer. After visualization with an avidin-biotin alkaline phosphatase procedure, the blot is post-stained with India ink to visualize the protein pattern context. To sequence spots, proteins are transferred to Immobilon, stained with Coomassie, and directly subjected to automated gas phase sequencing. The immunoblot procedure can detect less than 0.1 pmol protein, and the sequencing procedure can detect less than 10 pmol protein. After transfer enough material remains on the gels to allow subsequent autoradiography or silver stain. Quantitative analysis of 2-D gels is examined in a companion paper. These procedures should enhance the utility of 2-D gels in neurochemical studies.
Mobbs
C
C
Rockefeller University, New York, NY 10021.
Berman
J
J
Marquardt
M
M
Pfaff
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004587:Electrophoresis, Agar Gel; D005260:Female; D007158:Immunologic Techniques; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D014697:Ventromedial Hypothalamic Nucleus / Q000032:analysis*
D009419:Nerve Tissue Proteins