Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap.
The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.
D000942:Antigens, Bacterial / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D004563:Electrochemistry / Q000295:instrumentation; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008970:Molecular Weight; D009177:Mycoplasma pneumoniae / Q000032:analysis / Q000276:immunology; D013194:Staining and Labeling
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008565:Membrane Proteins