Membrane protein subunit fractionation by means of inverse pore gradient elution polyacrylamide gel electrophoresis.
We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of a membrane preparation of Torpedo electric organ (2 mg of total protein) on short gels (2 cm) with linear or concave inverse pore gradients. All of the four subunits are isolated in homogeneous fractions of approx 70 micrograms/ml within only 4 h. Compared to the standard method, this means a 50% reduction in separation times with threefold higher concentrated protein fractions. We also give the theoretical explanation and experimental validation of the improved features resulting from short inverse-gradient polyacrylamide gels.
Roth
U
U
Institute of Physiological Chemistry, Johannes Gutenberg-University Mainz, Germany.
Schönfeld
G
G
Schröder
B
B
Schrattenholz
A
A
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005069:Evaluation Studies as Topic; D007526:Isoelectric Point; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D008962:Models, Theoretical; D008970:Molecular Weight; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000302:isolation & purification*; D014101:Torpedo
D008565:Membrane Proteins; D011978:Receptors, Nicotinic