Development of a microfluidics-based gel protein recovery system.
A PMMA microfluidic chip, in which fluid is manipulated to transport protein from a PAGE gel piece to a collection reservoir via a microfluidic channel, has been developed. The protein sample is mobilized out of the gel (loaded in a chip access hole) into a low EOF-CE microfluidic channel under the influence of an electric field. Simultaneously, hydrostatic pressure from the filled buffer reservoirs is used to direct the protein sample to a third reservoir, through a field-free channel connected to the electrophoresis channel. Using this novel process of protein transport from a gel sample, proteins from Coomassie-stained gels have been transferred into solution in 15-30 min, with good sample recovery, using a run buffer containing an anionic acid-labile surfactant. A variety of small- and medium-sized proteins were successfully recovered and detected using both electrospray and MALDI MS over gel loads of 0.1-10 microg. This technological tool is very important for extracting quality intact protein samples from polyacrylamide gels, from which accurate protein molecular weights and protein sequences can be obtained using intact molecules.
Razunguzwa
Trust Tariro
TT
Protea Biosciences, Morgantown, WV 26505, USA. trust.razunguzwa@proteabio.com
Biddle
April
A
Anderson
Heather
H
Zhan
Dongliang
D
Powell
Matthew
M
D053841:Electroosmosis / Q000379:methods*; D006874:Hydrostatic Pressure; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D019904:Polymethyl Methacrylate / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D011506:Proteins; D019904:Polymethyl Methacrylate