Browsing by Subject "Polyacrylamide gel electrophoresis"
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Item Amperometric DNA sensing using wired enzyme based electrodes(2003) Zhang, Yongchao; Heller, AdamA water soluble copolymer of acrylamide and 4-vinylpyridine complexed with [Os(bpy)2Cl]+/2+ (bpy = 2,2’-bipyridine), was synthesized. An electrodeposition method of making redox polymer films on electrodes was developed. This method was also shown to be effective in incorporating enzymes and amine-terminated DNA sequences in the redox polymer film. A 38-base DNA sequence was detected at 20 pM concentration in 15-35 μL droplets by an electrochemical enzyme-amplified sandwich-type assay on a mass-manufacturable screen printed carbon electrode with a diameter of 3.5 mm. A DNA-capturing oligonucleotide was attached to the pre-deposited redox polymer film using the electrodeposition method. The electrode was exposed to the droplet containing the tested DNA sample, and was then treated with a droplet containing horseradish peroxidase-labeled detection sequence. Formation of the capture-target-detection sandwich brought the horseradish peroxidase-label of the detection sequence in electrical contact with the redox polymer, making the sandwich an electrocatalyst for the reduction of hydrogen peroxide to water at + 0.2 V (Ag/AgCl). The radial diffusion of electrons through the redox polymer film on the microelectrode allowed the electrodeposition of a thicker film of the redox polymer, an increase in the loading of the capture sequence, and increased the collection efficiency of the electron vacancies originating in the electroreduced H2O2. With a 10-μm diameter carbon fiber microelectrode, as few as 3000 copies of the 38-basse DNA sequence were detected at 0.5 fM concentration in a 10 μL sample. A biofuel cell operating at a power density of 50 μW cm–2 at 0.5 V under physiological conditions (air saturated, pH 7.4, 0.14 M NaCl, 37.5°C, 15 mM glucose) was developed. The cell had a glucose electro-oxidizing anode and an O2 electro-reducing cathode. The anode and the cathode were 7 μm diameter, 2 cm long carbon fibers, on which the catalytic enzyme-redox polymer adducts were cross-linked. When the miniature cell operated at 0.5 V, the power output dropped to about 60% of its initial value after 2 days of continuous operation at 37.5°C.Item Central cell incongruity as a barrier to introgression in (Allium cepa L. x A. fistulosum L.) x A. cepa backcross F1BC3 generations(Texas Tech University, 2001-08) Mangum, Paul D.Polyacrylamide gel electrophoresis (PAGE) was used to study the inheritance of esterase isozyme (EST) phenotypes in Allium fistulosum L. Three A. fistulosum specific loci were identified from the most anodal zones of acitivity, EST-1, EST-4, EST-6. Each locus was found to have two alleles. Both EST-1 and EST-4 encode a null allele. Plants homozygous for the nuli atieies lacked expression of the respective band on the PAGE gel. Starch gel electrophoresis was used to study the inheritance of the Phosphoglucoisomerase (PGI) phenotype. Pgi-1 in A. fistulosum has two alleles, Pgi-1^, and Pgi-1'*. Goodnessof-fit of these alleles to Mendelian ratios was tested in an F2 population with the chi-square statistic. Linkage analysis was also conducted befr.vscn the new alleles presented in this report and with previously identified alleles of ADH, 6-PGDH, and PGM. An incongruity model is presented as a possible expiaiiaiiun for an anomaly in the germination and establishment percentages, as well as a distorted isozyme segregation pattern, observed in F2BG3 seed of an A. cepa x A. fistulosum interspecific hybrid. The F1BG3 parent expressed Pgi-1^, PGI alleles of both species; Pgi-1^ coming from A. cepa (Ac) and Pgi-1^ allele from A. fistulosum (Af). Data collected on the F2BG3 progeny population were percent germination and establishment, segregation of Ac and Af Pgi-1 alleles, and viability and genotype of non-germinated seed, seed that was not planted, and isolated embryo and endosperm tissue. Seed, embryo, and endosperm tissues exhibited 100% viability, despite lower germination and establishment peicentayes.. A pooled goodness-of-fit test of the segregation of Pgi-1 alleles in the populations to the expected Mendelian 1:2:1 ratio using the chi-square statistic gave a x^ = 185.9, well beyond the accepted limits at 2 degrees of freedom, and the 1:2:1 ratio was rejected. Another pooled chi-square goodness-of-fit test of the segregation of Pgi-1 alleles in the populations to a 1:1 ratio based on the incongruity model gave a x^ = 0.203, within the accepted limits at 2 degrees of freedom and the 1:1 ratio was not rejected. Chi-square tests of data from other populations reporting similar to PGI isozyme distortion suppoit the hypothesis.Item Characterization of extracellular neuraminidases produced by different group B streptococcal serotypes(Texas Tech University, 1986-05) Brown, Jacqueline G.Not availableItem Improving figures of merit and expanding applications for inductively coupled plasma mass spectrometry(2010-08) Finley-Jones, Haley Joy; Holcombe, James A.; Brodbelt, Jennifer S.; Crooks, Richard M.; Willets, Katherine A.; Browning, Karen S.; Sparks, Chris M.Although inductively coupled plasma mass spectrometry (ICP-MS) is generally considered a reliable analytical technique, increasing demands on its capabilities require continued research and improvements. ICP-MS is susceptible to both matrix effects and drift, leading to a decline in accuracy and precision. A number of techniques are routinely used to compensate for these issues. Internal standardization is one such solution that requires relatively simple sample preparation and yet offers the possibility of improving both accuracy and precision. In order to be effective, an optimal analyte/internal standard pair must be chosen. Traditionally, analyte/internal standard pairs are chosen based on similarities in mass and/or ionization potential. The present studies sought to develop a program that determined standards based on the minimization of analytical error. 102 masses were monitored over 27 perturbations, i.e., changes to sample matrix and operating parameters. The standard deviations of the analyte/internal standard ratios were then used as a measure of internal standard performance. A thorough statistical analysis was conducted to determine trends between a good analyte/internal standard pair and similarities in chemical property. Similarities in mass offered the strongest relationship to a good internal standard choice, although many exceptions existed. The program was then tested over time and multiple instrument optimizations as well as on a completely different ICP-MS instrument. Results of these tests suggest that the data originally collected for the prediction program is not instrument-specific and thus provided a broader base of useful applications. Due to its unmatched sensitivity and multielement capabilities, ICP-MS is frequently utilized for biological samples. A more recent application, however, seeks to use ICPMS for the purpose of determining specific associations between metals and proteins. Such speciation requires a high resolution and reproducible separation prior to ICPMS analysis. Gel electrophoresis offers good separation and is well matched with the scanning properties of laser ablation sample introduction. The present study utilized native gel electrophoresis coupled with a uniquely modified electroblot system to improve sensitivity and to elucidate additional information. Chemically modified quartz fiber filters were successfully used as the transfer membrane to improve protein and metal capture efficiency.Item The use of protein gel electrophoresis for the detection of host plant resistance in cotton to the root-knot nematode(Texas Tech University, 1986-08) Smith, Roger GaryThirty-two germplasm sources of Gossypi um hi rsutum L. were inoculated with 1,000 larvae of Meloidogyne incognita (Chitwood) for six days. Root segments were excised and electrophoretically separated for protein content. Results show that resistant cotton plants respond to infection by root-knot nematodes quicker and more intensely than susceptible cotton plants. The most common type of protein band variation observed was in the darkness and thickness of various bands, and individual bands did not change uniformly among germplasms. These results indicate that polyacrylamide gel electrophoresis can be used to eliminate cotton plants which are susceptible to the root-knot nematode and can become a practical means to enhance the development of nematode resistant cultivars on the Texas High Plains.