Analysis of protein expression and compartmentalization in retinal neurons using serial tangential sectioning of the retina.
The progress in understanding visual signal transduction in vertebrate photoreceptors, arguably the best studied G protein-mediated signal transduction cascade in modern biology, was facilitated by the unique anatomy of rod photoreceptors. Held only by thin connected cilia, rod outer segments can be readily separated from the rest of the retina simply by shaking, and then purified by gradient centrifugation. The availability of such an efficient procedure of rod outer segment purification not only previously facilitated the identification of many principal visual signaling proteins located in this cellular compartment, but it is also currently being exploited in proteomics studies. In this paper, we describe a simple and inexpensive technique that allows for the quantitative analysis of protein expression within different subcellular compartments of photoreceptors, and could also be used for studying protein expression in the secondary retinal neurons. This technique is based on the Western blot analysis of the protein content of serial sections obtained by tangential sectioning of flat-mounted frozen retinas from mouse and rat, and it could serve as a way to validate proteomic data, similar to the way the quantitative RT-PCR technique is used for validation of gene-microarray data. To demonstrate the utility of this technique, we have determined the expression profiles in normal mouse retina of several signaling, energy-producing, and chaperone proteins, which were recently identified in bovine rod photoreceptors by mass spectrometry.
Song
Hongman
H
Departments of Ophthalmology and Biochemistry, West Virginia University School of Medicine and West Virginia University Eye Institute, Morgantown, West Virginia 26506.
Sokolov
Maxim
M
D000818:Animals; D002417:Cattle; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012160:Retina / Q000378:metabolism*; D055351:Retinal Neurons / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D015398:Signal Transduction
D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D020543:Proteome; D017868:Cyclic AMP-Dependent Protein Kinases; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins