Neurochemical Micromethods

  • Volker Neuhoff


This paper presents some details of refined micromethods employed in the neurochemical analysis of single neurons and sub-cellular structures and gives some examples of results obtained with these methods.


Water Soluble Protein Helix Pomatia Disc Electrophoresis Swing Bucket Rotor Beef Brain 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Althaus, H.-H., G. Briel, W. Dames, and V. Neuhoff: Zelluläre und molekulare Grundlagen der nervösen Erregungs-speicherung. Neurochemi sehe Mikroanalysen des Rückenmarks der Katze nach posttetanischer Potenzierung monosynaptischer Reflexe. In: Sonderforschungsbereich 33 Nervensystem und biologische Information, Göttingen 1969–1972, 107–121 (1972).Google Scholar
  2. Ansorg, R., W. Dames, and V. Neuhoff: Mikro-Disk-Elektrophorese von Hirnproteinen. II. Untersuchung verschiedener Extraktionsverfahren. Arzneimittelforschung 21, 699–710 (1971).Google Scholar
  3. Ansorg, R., and V. Neuhoff: Micro-disc electrophoresis of brain proteins. III. Heterogenity of the nervous specific protein S-100. Intern. J. Neuroscience 2, 151–160 (1971).CrossRefGoogle Scholar
  4. Brante, G.: Studies on lipids in the nervous system. Acta physic, scand. 18: Suppl. 63, 1–184 (1949).Google Scholar
  5. Brewer, Ü. M.: Artifact produced in disc electrophoresis by ammonium persulfate. Science 166, 256–257 (1967).CrossRefGoogle Scholar
  6. Briel, G., E. Gylfe, B. Hellmann, and V. Neuhoff: Micro-determination of free amino acids in pancreatic islets isolated from obese-hyperglycemic mice. Acta Phys. Scand. 84, 247–253 (1972).CrossRefGoogle Scholar
  7. Briel, G., and V. Neuhoff: Microanalysis of amino acids and there determination in biological material using dansylchloride. Hoppe-Seyler’s Z. Physiol. Chem. 353, 540–553 (1972).CrossRefGoogle Scholar
  8. Briel, G., V. Neuhoff, and N. N. Osborne: Determination of amino acids in single identifiable nerve cells of Helix pomatia. Intern. J. Neuroscience 2, 129–136 (1971).CrossRefGoogle Scholar
  9. Calissano, P., B. W. Moore, and A. Friesen: Effect of calcium ion on S-100, a protein of the nervous system. Biochemistry 8, 4318–4326 (1969).CrossRefGoogle Scholar
  10. Casola, L., H. Weise, and V. Neuhoff: In vitro protein synthesis by optic nerves. Hoppe-Seyler’s Z. Physiol. Chem. 350, 1175 (1969).Google Scholar
  11. Catsimpoolas, N.: Micro isoelectric focussing in Polyacrylamide gel columns. Anal. Biochem. 26, 480–482 (1968).CrossRefGoogle Scholar
  12. Choules, G. L., and B. H. Zimm: An acrylamide gel soluble in scintillation fluid. Its application to electrophoresis at neutral and low pH. Analyt. Biochem. 13, 336–344 (1965).CrossRefGoogle Scholar
  13. Cicero, T. J., and B. W. Moore: Turnover of the brain specific protein, S-100. Science 169, 1333–1334 (1970).CrossRefGoogle Scholar
  14. Cleveland, S., J. Haase, H.-G. Ross, and B. Vogel: Zelluläre und molekulare Grundlagen der nervösen Erregungs-speicherung, Motoneurone, post-tetanische Potenzierung und recurrente Inhibition. In: Sonderforschungsbereich 33 Nervensystem und biologische Information, Göttingen 1969–1972, 96–106 (1972).Google Scholar
  15. Cremer, Th., W. Dames, and V. Neuhoff: Micro disc electrophoresis and quantitative assay of glucose-6-phosphate dehydrogenase at the cellular level. Hoppe-Seyler’s Z. Physiol. Chem. 353, 1317–1329 (1972).CrossRefGoogle Scholar
  16. Curtis, D. R., and J. C. Eccles: Synaptic action during and after repetitive stimulation. J. Physiol. 150, 374–398 (1960).Google Scholar
  17. Dale, G., and A. L. Latner: Isoelectric focussing in Poly-acrylamide gels. Lancet 1, 847–848 (1968).CrossRefGoogle Scholar
  18. Dames, W., H. R. Maurer, and V. Neuhoff: Micro antigen-antibody crossed electrophoresis in vertical agarose gels following micro-disc electrophoresis. Hoppe-Seyler’s Z. Physiol. Chem. 353, 554–558 (1972).CrossRefGoogle Scholar
  19. Dames, W., V. Neuhoff, and Th. Cremer: Mikroelektrophoretische Trennung und Bestimmung von Dehydrogenasen einzelner Zellen. Naturwissenschaften 59, 126 (1972).CrossRefGoogle Scholar
  20. Dannies, P. S., and L. Levine: Demonstration of subunits in beef brain acidic protein (S-100). Biochem. biophys. Res. Commun. 37, 587–592 (1969).Google Scholar
  21. Davies, B. J.: Disc-electrophoresis II. Method an Application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404–427 (1964).CrossRefGoogle Scholar
  22. Eccles, J., and A. K. Mcintyre: The effect of disuse and of activity on mammalian spinal reflexes. J. Physiol. 121, 492–516 (1953).Google Scholar
  23. Fantes, K. H., and A. G. S. Furminger: Proteins, persulphate and disc electrophoresis. Nature 215, 750–751 (1967).CrossRefGoogle Scholar
  24. Filipowicz, W., G. Vincendon, P. Mandel, and G. Gombos: Topographical distribution of fast and slow migrating fractions of beef brain S-100. Life Science 7, 1243–1250 (1968).CrossRefGoogle Scholar
  25. Gombos, G., W. Filipowicz, and G. Vincendon: Fast and slow components of S 100 protein fraction: regional distribution in bovine central nervous system. Brain Research 26, 475–479 (1971a).Google Scholar
  26. Gombos, G., G. Vincendon, J. Tardy, and P. Mandel: Hétérogénéité électrophorétique et préparation rapide de la fraction protéique S-100. C.R. Acad. Sci. Paris 263, 1533–1535 (1966).Google Scholar
  27. Gombos, G., J.-P. Zanetta, P. Mandel, and G. Vincendon: Studies of F-SI00, a neurospecific protein fraction. I. In vivo presence of fast and slow migrating components of S-100 protein fraction. Biochimie 53, 635–644 (1971b).CrossRefGoogle Scholar
  28. Gombos, G., J.-P. Zanetta, P. Mandel, and G. Vincendon: Studies on F-S100, a neurospecific protein fraction: I. Molecular heterogeneity of S-100 protein fraction. Biochimie 53, 645–655 (1971c).CrossRefGoogle Scholar
  29. Gray, W. R., and B. S. Hartley: The structure of a chymotryptic peptide from pseudomonas cytochrome c-551. Biochem. J. 89, 59 P, 379–380 (1963).Google Scholar
  30. Grossbach, U.: Acrylamide gel electrophoresis in capillary columns. Biochem. Biophys. Acta 170, 180–182 (1965).Google Scholar
  31. Hyden, H.: Quantitative assay of compounds in isolated fresh nerve cells and glial cells from control and stimulated animals. Nature, Lond. 184, 433–435 (1959).CrossRefGoogle Scholar
  32. Hyden, H., K. Bjurstam, and B. Mc Ewen: Protein separation at the cellular level by micro disc electrophoresis. Annal. Biochem. 17, 1–15 (1966).CrossRefGoogle Scholar
  33. Kandel, E. R., and W. A. Spencer: Cellular neurophysiological approaches in the study of learning. Physiol. Rev. 48, 65–134 (1968).Google Scholar
  34. King, E. E.: Disc electrophoresis: avoiding artifacts caused by persulfate. J. Chromatg. 53, 559–563 (1970).CrossRefGoogle Scholar
  35. Kolin, A.: Separation and concentration of proteins in a pH field combined with an electric field. J. Chem. Phys. 22, 1628–1629 (1954).CrossRefGoogle Scholar
  36. Lloyd, D. P. C.: Posttetanic potentiation of respone in mono-synaptic reflex pathways of the spinal cord. J. gen. Physiol. 33, 147–170 (1949).CrossRefGoogle Scholar
  37. Maurer, H. R.: Disc electrophoresis and related techniques of Polyacrylamide gel electrophoresis. In: Working Methods in Modern Sciences ( K. Fischbeck, ed.), Walter de Gruyter, Berlin, New York (1971).Google Scholar
  38. Mc Ewen; B., and H. Hyden: Study of specific brain proteins on the semi-micro scale. J. Neurochem. 13, 823–833 (1966).CrossRefGoogle Scholar
  39. Moore, B. W.: A soluble protein characteristic of the nervous system. Biochem. biophys. Res. Commun. 19, 739–744 (1965).Google Scholar
  40. Moore, B. W., and V. J. Perez: Specific acidc proteins of the nervous system. In: Physiological and Biochemical Aspects of Nervous Integration (F. D. Carlson, ed.), pp. 343–359, Prentice Hall, (1968).Google Scholar
  41. Neuhoff, V.: “feedback-Neurone” im Zentralnervensystem des Menschen. Naturwissenschaften 54, 287–288 (1967).Google Scholar
  42. Neuhoff, V: Micro-Disc-Electrophorese von Hirnproteinen. Arzneimittelforschung 18, 35–39 (1968a).Google Scholar
  43. Neuhoff, V.: Simplified technique of high-speed capillary centrifugation. Analytical Biochemistry 23, 359–362 (1968b).CrossRefGoogle Scholar
  44. Neuhoff, V.: Einfaches Verfahren zur hochtourigen Kapi11a-renzentrifugation. GIT 13, 86–87 (1969).Google Scholar
  45. Neuhoff, V.: Manual, 1st EMBO-Course on Micromethods in Molecular Biology, Max-Planck-GesellSchaft, Dokumenta-tionsstelle (1970).Google Scholar
  46. Neuhoff, V.: Micromethods in Molecular Biology, Springer-Verlag, Heidelberg, New York in press (1972).Google Scholar
  47. Neuhoff, V.: Manual, 2nd EMBO-Course on Micromethods in Molecular Biology, Max-Planck-Gesellschaft, Dokumen-tationsstelle (1971a).Google Scholar
  48. Neuhoff, V.: Wet weight determination in the lower milligram range. Analytical Biochemistry 270–271 (1971b).Google Scholar
  49. Neuhoff, V., G. Briel, and A. Maelicke: Characterization and microdetermination of histidine as its dansyl-compounds. Arzneimittelforschung 21, 104–107 (1971).Google Scholar
  50. Neuhoff, V., F. Von Der Haar, E. Schlimme, and E. Weise: Zweidimensionale Chromatographie von Dansyl-Aminosäuren im pico-Mol-Bereich, angewandt zur direkten Charakterisierung von Transfer-Ribonucleinsäuren. Hoppe-Seyler’s Z. Physiol. Chem. 350, 121–123 (1969).CrossRefGoogle Scholar
  51. Neuhoff, V., and F. Kiehl: Dialysiergeräte für Volumen zwischen 10 und 500 ul. Arzneimittelforschung 19, 1898–1899 (1969).Google Scholar
  52. Neuhoff, V., and A. Lezius: Nachweis der Substruktur von DNA-Polymerasen, der enzymatisch aktiven Proteinkomponente und ihrer Enzym-Substrat-Komplexe mit der Micro-Disc-El ectrophorese. Hoppe-Seyler’s Z. für Physiol. Chem. 348, 1239 (1967).Google Scholar
  53. Neuhoff, V., and A. Lezius: Nachweis und Charakterisierung von DNS Polymerasen durch Micro-Disc-Electrophorese. Z. Naturforschung 23b, 812–819 (1968).Google Scholar
  54. Neuhoff, V., B. Mühlberg, and J. Meier: Strom- und spannungs-konstantes Netzgerät für die Micro-Disc-Electrophorese. Arzneimittelforschung 17, 649–651 (1967).Google Scholar
  55. Neuhoff, V., D. Müller, and V. Ter Meulen: Präparation von Ganglienzellen für cytophotometrische Untersuchungen. Z. Wiss. Mikroskopie 69, 2, 65 - 72 (1968).Google Scholar
  56. Neuhoff, V., and W.-B. Schill: Kombinierte Mikro-Disk-Elektrophorese und Mikro-Immunpräzipitation von Proteinen. Hoppe-Seyler’s Z. Physiol. Chem. 349, 795–800 (1968).CrossRefGoogle Scholar
  57. Neuhoff, V., W.-B. Schill, and D. Jacherts: Nachweis einer RNA-abhängigen RNA-Replicase aus immunologisch kompetenten Zellen durch Mikro-Disk-Elektrophorese. Hoppe-Seyler’s Z. Physiol. Chem. 351, 157–162 (1970).CrossRefGoogle Scholar
  58. Neuhoff, V., W.-B. Schill, and H. Sternbach: Microanalysis of pure deoxyribonucleic acid-dependent rubonucleic acid polymerase from Escherichia coli. Biochem. J. 117, 623–631 (1970).Google Scholar
  59. Neuhoff, V., and M. Weise: Determination of picomole quantities of y-amino-butyric-acid (GABA) and serotonin. Arzneimittelforschung 20, 368–372 (1970).Google Scholar
  60. Neuhoff, V., H. Weise, and H. Sternbach: Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. VI. Determination of amino acid composition. Hoppe-Seyler’s Z. Physiol. Chem. 351, 1395–1401 (1970).CrossRefGoogle Scholar
  61. Ornstein, L.: Disc-electrophoresis I. Background and theory. Ann. N.Y. Acad. Sci. 121, 321–349 (1964).CrossRefGoogle Scholar
  62. Osborne, N. N.: Effect of electrical stimulation on the in vivo metabolism of glucose and glutamic acid in an identified neuron. Brain Research 41, 237–241 (1972b).CrossRefGoogle Scholar
  63. Osborne, N. N.: The in vivo synthesis of serotonin in an identiefied serotonin-containing neuron of Helix pomatia. Intern. J. Neuroscience 3, 215–228 (1972a).CrossRefGoogle Scholar
  64. Osborne, N. N., H.-H. Althaus, and V. Neuhoff: Phospholipids in the nervous system of the gastropod mollusc Helix pomatia, and the in vivo incorporation of 32P into the phospholipids of identified neurons. Comp. Biochem. Physiol. (1972), in press.Google Scholar
  65. Osborne, N. N., R. Ansorg, and V. Neuhoff: Micro-disc electrophoretic separation of soluble proteins from nervous and other tissues of Helix (Pulmonate mollusca). Intern. J. Neuroscience 1, 259–264 (1971).CrossRefGoogle Scholar
  66. Osborne, N. N., G. Briel, and V. Neuhoff: Distribution of GABA and other amino acids in different tissues of the gastropod mollusc Helix pomatia, including in vitro experiments with 14C glucose and 14C glutamic acid. Internat. J. Neuroscience 1, 265–272 (1971).CrossRefGoogle Scholar
  67. Osborne, N.N. and G. A. Cottrell: Amine and amino acid micro-analysis of two identified snail neurons with known characteristics. Experientia 28, 656–658 (1972).CrossRefGoogle Scholar
  68. Osborne, N. N., and V. Neuhoff: Neurochemical studies on characterized neurons. Naturwissenschaften (1972), in press. (1972).Google Scholar
  69. Osborne, N. N., B. Powell, and G. A. Cottrell: The effect of electrical stimulation on the levels of free amino acids and related compounds in the snail brain. Brain Res. 41, 379–386 (1972).CrossRefGoogle Scholar
  70. Petzold, U., W. Dames, G. H. M. Gottschewski, and V. Neuhoff: Das Proteinmuster in frühen Entwicklungsstadien des Kaninchens. Cytobiologie 5, 272–280 (1972).Google Scholar
  71. Pun, J. Y., and Lombrozo: Microelectrophoresis of brain and pineal protein in Polyacrylamide gel. Annal. Biochem. 9, 9–20 (1964).CrossRefGoogle Scholar
  72. Quentin, C.-D., and V. Neuhoff: Micro-isoelectric focussing for the detection of LDH isoenzymes in different brain regions of rabbit. Intern. J. Neuroscience 4, 17–24 (1972).CrossRefGoogle Scholar
  73. Riley, R. F., and M. K. Coleman: Isoelectric fractionation of proteins on a microscale in Polyacrylamide and agarose matrices. J. Lab. and Clin. Med. 72, 714–720 (1968).Google Scholar
  74. Rüchel, R.: Mikroelektrophoresen von RNS-Basen, Anwendung zur Untersuchung bestimmter Hirnregionen und kritische Analyse der Methode. Inauguraldissertation, Göttingen (1971).Google Scholar
  75. Schiefer, H.-G., and V. Neuhoff: Fluorometric determiantion of phospholipids on the cellular level. Hoppe-Seyler’s Z. Physiol. Chem. 352, 913–926 (1971).CrossRefGoogle Scholar
  76. Sear, P. G., and B. Roizman: An improved procedure for H3 14 and C counting in acrylamide gels with an nonaqueous scintillation system. Analyt. Biochem. 26, 197–200 (1968).CrossRefGoogle Scholar
  77. Seiler, N.: Methods of biochemical analysis. Use of the Dansyl reaction in biochemical analysis. In: Interscience Publ. (D. Glick, ed.), Vol. 18, 259–337, John Wiley amp; Sohn, New York, London, Sydney, Toronto (1970).Google Scholar
  78. Svensson, H.: Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients: I. The differential equation of stae of solute concentrations at a steady state and its solution for simple cases. Acta Chem. Scand. 15, 325–341 (1961).CrossRefGoogle Scholar
  79. Tardy, J., K. Uyemura, G. Gombos, and G. Vincendon: Caractêrisation dun groupe de proteins spécifiques du système nerveux. J. Physiol.59, 510 (1967).Google Scholar
  80. Ter Meulen, V., D. Moller, G. Enders-Rockle, V. Neuhoff, M. Y. Käckell, and G. Joppich: Ist die subakute progressive Panenzephalitis eine Masernerkrankung? Dtsch. Med. Wschr. 93, 1303–1308 (1968).CrossRefGoogle Scholar
  81. Ter Meulen, V., D. Müller, V. Neuhoff, and G. Joppich: Immunhistological, microscopcial and neurochemical studies on encephalitides. V. Subacute sclerosing panencephalitis. Cytophotometric studies on isolated nerve cells. Acta Neuropath. 15, 128–141 (1970).CrossRefGoogle Scholar
  82. Uyemura, K., J. Tardy, G. Vincendon, P. Mandel, and G. Gombos: Mise en évidence de protéines spécifiques du cerveau chez les Mammifêeres. C.R. Soc. Biol. 161, 1396–1399 (1967).Google Scholar
  83. Uyemura, K., G. Vincendon, G. Gombos, and P. Mandel: Purification and some properties of S-l00 protein fractions from sheep and pig brains. J. Neurochem. 18, 429–438 (1971).CrossRefGoogle Scholar
  84. Vesterberg, O., and H. Svenssen: Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients: IV. Further studies on the resolving power in connection with separation of myoglobins. Acta Chem. Scand. 20, 820–834 (1966).CrossRefGoogle Scholar
  85. Vincendon, G., A. Waksman, K. Uyemura, J. Tardy, and G. Gombos: Ultracentirfugal behavior of beef brain S-100 protein fraction. Arch. Biochem. Biophys. 120, 233–235 (1967).CrossRefGoogle Scholar
  86. Woods, K. R., and K. T. Wang: Separation of danysl-amino acids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369–370 (1967).Google Scholar
  87. Wrigley, C. W.: Analytical fractionation of plant and animal proteins by gel electrofocussing. J. Chromatog. 36, 362–365 (1968).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1973

Authors and Affiliations

  • Volker Neuhoff
    • 1
  1. 1.Arbeitsgruppe NeurochemieMax-Planck-Institut für experimentelle MedizinGöttingenGermany

Personalised recommendations