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1.
Electrophoresis ; 40(5): 817-823, 2019 03.
Article in English | MEDLINE | ID: mdl-30556148

ABSTRACT

With the growth of the biopharmaceutical industry, there is a need for rapid size-analysis of proteins on the megaDalton scale. The large pore sizes needed for such separations cannot be easily reached by pushing the current limits of size-exclusion chromatography or gel electrophoresis. The concept detailed here is the formation of arbitrarily wide pores by packing nonporous colloidal silica in capillaries. This method can be called packed-capillary electrophoresis, or "pCE". Electrophoresis of protein standards (11-155 kDa) by pCE, using 345 nm diameter particles in 100 µm diameter capillaries, gives 2x higher resolution than a typical PAGE gel in 1/6 of the time. The electropherograms show that pCE is highly efficient, with half-micrometer plate heights for all seven standards, giving 105 plates for a 50 mm length. The large pore radius of 65 nm enables baseline resolution of proteins of 0.72, 1.048 and 1.236 MDa in less than 15 min. The short separation time of pCE is attributed to the absence of small pores that restrict protein migration in gels. The pCE separation is applied to the analysis of a stressed pharmaceutical-grade IgG4 sample, giving unprecedented baseline resolution of monomer, dimer, trimer and tetramer in less than 10 min.


Subject(s)
Colloids/chemistry , Electrophoresis, Capillary/methods , Recombinant Proteins , Silicon Dioxide/chemistry , Particle Size , Porosity , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
2.
Anal Chem ; 85(15): 7102-8, 2013 Aug 06.
Article in English | MEDLINE | ID: mdl-23796076

ABSTRACT

The enzyme-linked immunosorbent assay is commonly used for research and clinical applications but typically suffers from a limited linear range and is difficult to multiplex. The fluorophore-linked immunosorbent assay is a closely related technique with good linear range and the ability to detect multiple antigens simultaneously but is typically less sensitive. Here, we demonstrate a near-infrared, surface-enhanced fluorophore-linked immunosorbent assay with sensitivity comparable to its enzyme-linked counterpart. A 59-fold enhancement to sensitivity (slope of linear fit) and an 8-fold improvement in LOD are demonstrated on a direct assay with rabbit immunoglobulin-G as a model system. The technique is also tested on a clinically relevant assay to detect alpha-fetoprotein, in which a 42-fold enhancement to sensitivity is demonstrated along with a 16-fold improvement in LOD. The technique enables these accomplishments while maintaining the entire traditional assay protocol and simply adding two steps at the end. This technique may prove superior to current protocols for biomarker research and clinical diagnoses, which require high sensitivity along with quantitation over an extended range.


Subject(s)
Fluorescent Dyes/chemistry , Immunosorbent Techniques , Infrared Rays , Animals , Benzenesulfonates/chemistry , Immunoglobulin G/immunology , Indoles/chemistry , Spectrometry, Fluorescence , Surface Properties , alpha-Fetoproteins/analysis
3.
Nucleic Acids Res ; 36(18): e121, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18723573

ABSTRACT

Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis.


Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA/biosynthesis , Sequence Analysis, DNA , Streptavidin/chemistry , Biotinylation , Catalysis , DNA/chemistry , DNA-Directed DNA Polymerase/metabolism , Freezing , Kinetics , Protein Engineering , Temperature , Templates, Genetic
4.
J Biomol Tech ; 18(3): 147-9, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17595310

ABSTRACT

We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%.


Subject(s)
DNA Primers/chemistry , Directed Molecular Evolution/methods , Mutagenesis, Site-Directed , DNA Primers/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics
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