Extraction of proteins within ultrathin-layer polyacrylamide electrophoresis (SDS-PAGE) and isoelectric focusing (PAGIF) of cryostat sections and tissue culture specimens.
Separations on a micro- and ultramicroscale by electrophoresis and isoelectric focusing of protein quantities between 10(-6) and 10(-12) g have notable interest, particularly in cytology and histology. We performed the methods of ultrathin-layer polyacrylamide horizontal electrophoresis, isoelectric focusing and protein mapping with some modifications in order to study protein extraction and applied them for cryostat sectioned muscle tissues and singular dorsal root ganglia of the chicken in tissue culture. Further we established an extraction chamber for histological specimens. The basis for ultrathin-layer electrophoresis is the ultrathin (0.12 mm-0.36 mm) polyacrylamide gel on glass plates or microscopic slides. This method allows a considerable reduction of the amount of proteins at the range of 10(-9) g and is thus appropriate for direct extraction of proteins within electrophoresis (10 min, 5 mA 200 V) or isoelectric focusing (10 min, 10 mA, 500 V) of cryostat-sections (7-20 micron thickness) or tissue culture specimens. The advantages of these techniques for extraction of soluble proteins in immunohistochemistry as well as for handling to obtain optimal resolution (compared with electrophoresis of conventional extracted proteins) will be demonstrated.
Inczédy-Marcsek
M
M
Institut für Anatomie, Universität Regensburg, FRG.
Lindner
E
E
Hässler
R
R
Zwack-Megele
G
G
Roisen
F
F
Yorke
G
G
D000818:Animals; D002645:Chickens; D046508:Culture Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D005615:Freezing; D005727:Ganglia, Spinal / Q000032:analysis; D006652:Histological Techniques; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D008867:Microtomy; D011506:Proteins / Q000302:isolation & purification*
D011506:Proteins