Enhanced protein recovery after electrotransfer using square wave alternating voltage.
Protein identification is becoming a complement to the available fully sequenced genomes. To meet the challenge, newly developed techniques for high throughput protein identification using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and peptide mass fingerprint are needed. Two years ago, a parallel protein digestion process was proposed. It provided a collecting polyvinylidene difluoride (PVDF) membrane able to be scanned by MALDI. Acquired data were used to recreate a virtual multidimensional image. Voltage used during this protein electroblotting technique was an unusual square wave alternative voltage (SWAV). The goal of the current study is to evaluate quantitatively the efficiency of the SWAV compared with a classical electroblot process on intact proteins. The effect of the pulsed electric field and the buffer composition were compared to a standard continuous transblotting process defined as the gold standard. Combination of the pulsed asymmetric electric field with 3-(cyclohexylamino)-1-propane-sulfonique acid (CAPS) buffers showed an average 65% increase of protein recovery. Moreover, a strongest effect is observed for high M(r) proteins. In conclusion, the present study highlighted a positive influence of the "shaking" effect of the asymmetric alternative voltage on gel protein extraction.
Bienvenut
Willy V
WV
Department of Pathology, Central Clinical Chemistry Laboratory, Geneva University Hospital, Geneva, Switzerland. william.bienvenut@dim.hcuge.ch
Déon
Catherine
C
Sanchez
Jean-Charles
JC
Hochstrasser
Denis F
DF
D002021:Buffers; D004560:Electricity; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification*; D015203:Reproducibility of Results
D002021:Buffers; D011506:Proteins