Rapid electroelution of two-dimensionally separated protein mixtures: its use in in vitro assays of T cell activities.
A recently developed electroelution method for separated mixtures of proteins and its application in vaccine research were investigated. The method combines the high resolution power of two-dimensional gel electrophoresis with the advantage of direct probing of separated proteins with viable cells. An electroelution time of only 30 min was sufficient for complete protein transfer, as shown by Coomassie Brilliant Blue and silver staining. Inclusion of sodium dodecyl sulfate (SDS) into the electrophoresis buffer for the second dimension considerably improved the separation capacity. Furthermore, because of the low concentration of SDS (0.03%) no deleterious effects on the cells were seen. It was shown that T lymphocytes from cattle vaccinated with dead M. bovis BCG responded to numerous mycobacterial protein antigens, whereas unvaccinated control animals showed no, or very weak, responses. A comparison of T cell proliferation profiles obtained with different protein separations demonstrated the reproducibility of the method.
Gulle
H
H
Massey University, Palmerston North.
Kaufmann
S H
SH
Moriarty
K M
KM
D000818:Animals; D001500:BCG Vaccine / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D002455:Cell Division; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008213:Lymphocyte Activation; D009163:Mycobacterium bovis / Q000276:immunology; D011506:Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D013997:Time Factors
D001500:BCG Vaccine; D011506:Proteins; D012967:Sodium Dodecyl Sulfate