Micro-scale open-tube capillary separations of functional proteins.
This article describes a novel technique whereby fully functional proteins or multiprotein complexes are efficiently extracted from biological samples to chemically derivatized walls of fused-silica open-tube capillary columns. Proteins are eluted with very high yields into elution volumes that are smaller in volume than the internal volume of the open-tube capillary column itself, thereby achieving 100-fold increases in target protein concentrations from starting samples of less than 1 ml. The open-tube capillary columns are designed for single use; combined with the physical and chemical characteristics of the open-tube capillary column, this provides exceptional purity to the eluted proteins. Affinity-based open-tube capillary columns are demonstrated here to purify, enrich, and maintain functionality for a monomeric and dimeric enzyme, a low-abundance HeLa nuclear complex, and a light-harvesting octadecameric membrane protein complex. The design of the open-tube capillary column allows for facile direction of the processed protein sample to any number of final detection techniques and is capable of generating final protein concentrations required for many structural biology experiments. The open-tube capillary columns are also characterized by exceptional ease of use. Current designs allow for up to 10 open-tube capillary columns to be applied simultaneously with no fundamental impediments to even greater parallel operation.
Hanna
Christopher
C
PhyNexus Inc., San Jose, CA 95136, USA. christphanna@gmail.com
Gjerde
Douglas
D
Nguyen
Liem
L
Dickman
Mark
M
Brown
Paul
P
Hornby
David
D
D001426:Bacterial Proteins / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000295:instrumentation* / Q000379:methods*; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D004268:DNA-Binding Proteins; D005982:Glutathione Transferase / Q000302:isolation & purification; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000302:isolation & purification; D006801:Humans; D045342:Light-Harvesting Protein Complexes / Q000302:isolation & purification; D008832:Microchemistry / Q000295:instrumentation; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D034521:Nuclear Matrix-Associated Proteins / Q000302:isolation & purification; D050810:Octamer Transcription Factors / Q000302:isolation & purification; D038941:Polypyrimidine Tract-Binding Protein / Q000302:isolation & purification; D016601:RNA-Binding Proteins / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D045342:Light-Harvesting Protein Complexes; D046912:Multiprotein Complexes; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; C450006:PTBP1 protein, human; D016601:RNA-Binding Proteins; C094744:light-harvesting complex 1, Rhodospirillum; D038941:Polypyrimidine Tract-Binding Protein; D005982:Glutathione Transferase