| 1. |
- Björn, Lars Olof
(författare)
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Simple spectropolarimetric accessory for dual-wavelength spectrophotometers
- 1986
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X. ; 12:5-6, s. 355-358
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Tidskriftsartikel (refereegranskat)abstract
- A simple spectropolarimetry accessory for the Aminco DW-2a spectrophotometer is described. It is useful in the wavelength range 414–671 nm (with slight modifications 200–800 nm) and gives a resolution in optical rotation better than 0.01°. The principle should be applicable to many other types of spectrophotometers.
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| 2. |
- Bradoo, Sapna, et al.
(författare)
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Microwave-assisted rapid characterization of lipase selectivities.
- 2002
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X. ; 51:2, s. 115-120
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Tidskriftsartikel (refereegranskat)abstract
- A rapid screening procedure for characterization of lipase selectivities using microwaves was developed. The rate of reaction of various commercial lipases (porcine pancreas, Mucor miehei, Candida rugosa, Pseudomonas cepacia) as well as lipases from laboratory isolates-Bacillus stearothermophilus and Burkholderia cepacia RGP-10 for triolein hydrolysis was 7- to 12-fold higher in a microwave oven as compared to that by pH stat. The esterification of sucrose/methanol and ascorbic acid with different fatty acids was also achieved within 30 s in a microwave using porcine pancreas, B. stearothermophilus SB-1 and B. cepacia RGP-10 lipases. The relative rates and selectivity of the lipases both for hydrolytic and synthesis reactions remains unaltered. However, the rate of reaction was dynamically enhanced when exposed to microwaves. Microwave-assisted enzyme catalysis can become an attractive procedure for rapid characterization of large number of enzyme samples and substrates, which otherwise is a cumbersome and time-consuming exercise.
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| 3. |
- Hsiao, K. C., et al.
(författare)
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An artifact in measurements of in vivo light-induced absorbance changes
- 1983
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X. ; 8:4, s. 271-274
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Tidskriftsartikel (refereegranskat)abstract
- The effects of scattered actinic radiation on photomultipliers (Hamamatsu R-562) were investigated. Using cotton-wool to model dense biological preparations, it was found that the scattered actinic radiation received by the photomultiplier gives rise to phytochrome-like signals. This demonstrated the necessity to shield the photomultiplier from scattered actinic light for sensitive measurements with light-scattering preparations.
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| 4. |
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| 5. |
- Nahálková, Jarmila, et al.
(författare)
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Affinity analysis of lectin interaction with immobilized C- and O-gylcosides studied by surface plasmon resonance assay
- 2002
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 52:1, s. 11-18
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Tidskriftsartikel (refereegranskat)abstract
- A biosensor based on the surface plasmon resonance (SPR) principle was used for kinetic analysis of lectin interactions with different immobilized saccharide structures. A novel affinity ligands beta-D-glycopyranosylmethylamines derived from common D-aldohexoses linked to the carboxymethyl dextran layer of the SPR sensor surface served for interactions with a wide range of lectins. The method of preparation and use of the beta-D-mannopyranosyl glycosylated sensor surface was described. The results of affinity analysis of lectin-ligand interactions were evaluated and compared with data obtained from measurements using commercially available p-aminophenyl alpha-D-glycopyranosides. Possible applications and advantages of C- and O-glycosylated SPR biosensors are discussed.
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| 6. |
- Thorén, Sven A., et al.
(författare)
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A microcalorimetric vessel for monitoring cytotoxic reactions in the micro-submicrowatt region
- 1990
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X. ; 20:3, s. 217-225
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Tidskriftsartikel (refereegranskat)abstract
- A microcalorimetric vessel for monitoring the initial phases of cytotoxic reactions in the micro-submicrowatt region has been designed and tested. The vessel is intended to be used witha multichannel microcalorimetric system from Thermometric, Järfälla, Sweden and can be built up stepwise in a modular way. The different functions of perfusion, stirring and addition of small amounts of soluble immune reactants can be used separately but also in combination. This is accomplished by the use of a vertical stirring mechanism and a reaction vessel of low volume, 200 μl. The simplcity of the vessel permits an identical vessel to be used on the reference side, handled in parallel with the measuring vessel. No gas phase is present.
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| 7. |
- Haupt, Dan, et al.
(författare)
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Separation of (R)- and (S)-naproxen using micellar chromatography and an alpha 1-acid-glycoprotein column : application for chiral monitoring in human liver microsomes by coupled-column chromatography
- 1992
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 25:4, s. 273-284
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Tidskriftsartikel (refereegranskat)abstract
- A column-switching system for fast determination of (R)- and (S)-naproxen in liver microsomes has been developed. The centrifuged sample was injected directly onto a pre-column with octadecylcoated silica. The retained analytes were then directed to an alpha 1-AGP column using a mobile phase composed of phosphate buffer (pH 6.5), dimethylocytylamine (30 mM) and the nonionic surfactant, Tween 20 (40 g/l). The method gave high absolute recoveries and good repeatabilities: 99.6% (1.7% relative standard deviation) and 94.9% (2.4% R.S.D.) for the (R)- and (S)-naproxen, respectively. The use of a surfactant in combination with an aliphatic amine in the mobile phase involves reduced retention times with retained enantioselectivity. Furthermore, the presence of the surfactant makes it possible to inject biological samples directly into the chromatographic system.
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| 8. |
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| 9. |
- Lagerquist Hägglund, Christine, et al.
(författare)
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Centrifugal and chromatographic analyses of tryptophan and tyrosine uptake by red blood cells and GLUT1 proteoliposomes with permeability estimates and observations on dihydrocytochalasin B
- 2003
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 55:2, s. 127-140
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed transport into liposomes and proteoliposomes, separated the free and internalized radioactively labeled substrates by size-exclusion chromatography (SEC) and observed a net influx owing to nonfacilitated diffusion across the lipid bilayers during the separation. The permeabilities (10(-9) cm/s) of glucose transporter (GLUT1) proteoliposomes were estimated to be 4.6, 1.0, 1.4 and 2.1 for D-glucose, L-glucose, L-Tyr and L-Trp, respectively; 15, 3.3, 5.1 and 2.1 times higher than the corresponding permeabilities of liposomes. These values indicated that GLUT1 did not transport Tyr or Trp, or transported Tyr, and only Tyr, slowly. This interpretation was supported by further analyses. Dihydrocytochalasin B inhibited the transport of Tyr and, partially, Trp into human red blood cells (centrifugal analyses). It did not inhibit Tyr and Trp influx into GLUT1 proteoliposomes, but partitioned strongly into the bilayers and seemed to make them fragile. The GLUT1 inhibitor cytochalasin B and the GLUT1 substrate 2-deoxy-D-glucose did not inhibit Tyr transport into the cells. Upon immobilized biomembrane affinity chromatography, Trp decreased the cytochalasin B retardation by GLUT1 only at levels far above the physiological Trp concentration. Ethanol (commonly added to aqueous solutions for enhancing a compound's solubility) halved the retardation at 4% (v/v) concentration. Drastic modification of the SEC method is required to allow permeability measurements with nonlabeled and highly permeable substrates.
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| 10. |
- Nordstrom, T., et al.
(författare)
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Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing (TM) technology
- 2002
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 52:2, s. 71-82
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Tidskriftsartikel (refereegranskat)abstract
- A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing(TM) technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.
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| 11. |
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| 12. |
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| 13. |
- Södergren, E, et al.
(författare)
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Re-evaluation of the ferrous oxidation in xylenol orange assay for the measurement of plasma lipid hydroperoxides.
- 1998
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 37:3, s. 137-46
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Tidskriftsartikel (refereegranskat)abstract
- The ferrous oxidation in xylenol orange version 2 (FOX2) assay coupled with triphenylphosphine has recently been employed for the measurement of total plasma hydroperoxides (ROOHs). In this study, we have evaluated sample handling and the effect of storage conditions on ROOH levels in human plasma (n = 32). Mean level of ROOHs in fresh plasma was 8.35 +/- 3.09 mumol/l (range 4.03-19.5 mumol/l). Addition of butylated hydroxytoluene (BHT) immediately after sample collection had no effect on the concentration of ROOHs. Storage of samples at -70 degrees C for 6 weeks was associated with a variable degree of loss of detectable ROOHs. A mean ROOH level of 6.00 +/- 2.23 mumol/l (range 2.88-13.5 mumol/l) was recorded after 6 weeks of storage at -70 degrees C. There was no difference in the mean level of ROOHs between samples stored for 6 and 60 weeks at -70 degrees C. Inclusion of BHT had no effect on the stability of plasma ROOHs during prolonged storage. Intra-assay coefficients of variation were < 12%, with the lowest variation in fresh samples (7.6%). In conclusion, these results suggest that the FOX2 assay should be a useful tool for measurement of ROOHs in fresh plasma samples but not in stored samples.
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| 14. |
- Bagdonaite, Kristina, et al.
(författare)
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Analysis of 3-aminopropionamide: A potential precursor of acrylamide
- 2006
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Ingår i: Journal of Biochemical and Biophysical Methods (Proceedings). - : Elsevier BV. - 0165-022X. ; 69:1-2, s. 215-221
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Konferensbidrag (refereegranskat)abstract
- An analytical method for the analysis of 3-aminopropionamide (3-APA) based on derivatization with dansyl chloride and liquid chromatography/fluorescence detection was developed. We have analysed 3-APA formation in raw potatoes, grown and stored under different condition, green and roasted coffee beans and in freeze dried mixtures of asparagine with sucrose and glucose in molar ratio of 1:0.5, 1: 1, and 1: 1.5. In potatoes the 3-APA content varied depending on the potato variety. We detected 3-APA in potatoes up to 14 mu g/g fresh weight. In the model experiment glucose had a stronger capacity to form 3-APA. The substance was formed at temperatures as low as 130 degrees C. However, in the model experiment with sucrose 3-APA was formed not below 150 degrees C. In heated mixtures with increasing molar ratio of sucrose at 170 degrees C we noticed a decrease of 3-APA and in the same mixtures at 150 degrees C we observed an increase of 3-APA. In coffee 3-APA was not formed, neither in green nor in roasted beans. (c) 2006 Elsevier B.V. All tights reserved.
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| 15. |
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| 16. |
- Dolby, Viveka, et al.
(författare)
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Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor.
- 2004
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X. ; 58:3, s. 195-205
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Tidskriftsartikel (refereegranskat)abstract
- The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, Image-Phe7]-αMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 °C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.
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| 17. |
- Eriksson, Håkan
(författare)
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Controlled release of preservatives using dealuminated zeolite Y
- 2008
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:6, s. 1139-1144
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Tidskriftsartikel (refereegranskat)abstract
- This study demonstrates that dealuminated zeolite Y can act as a depot after adsorption of phenol derived preservatives. Upon suspension of zeolite loaded with the preservative m-cresol, equilibrium was quickly reached between free and adsorbed m-cresol. The equilibrium concentration of m-cresol was below 1 mM, however, it was enough to kill bacteria such as Escherichia coli and Staphylococcus aureus under metabolically active conditions. Killing of bacteria were not obtained under non-proliferating conditions and m-cresol was only released from the zeolite upon bacterial activity. Together, these results demonstrate an interesting potential use of dealuminated zeolite Y containing adsorbed preservatives for preventing microbial growth in numerous applications in industry and clinical setting.
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| 18. |
- Hakkarainen, Minna
(författare)
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Developments in multiple headspace extraction
- 2007
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:2, s. 229-233
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Forskningsöversikt (refereegranskat)abstract
- This paper reviews new developments in multiple headspace extraction (MHE), especially its combination with two miniaturized extraction techniques, solid-phase microextraction (SPME) and single-drop microextraction (SDME). The combination of the techniques broadens the applicability of SPME and SDME to quantitative determination of analytes in complex liquid and solid matrixes. These new methods offer several advantages over traditional liquid-solid, liquid-liquid and headspace extraction techniques. The potential applications include extraction of volatiles and semivolatiles from environmental and physiological samples and from different polymer products such as medical and biomedical materials, food packaging and building materials. The theoretical principals of the techniques are also briefly reviewed.
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| 19. |
- Hedin, Eva M. K., et al.
(författare)
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Low microwave-amplitude ESR spectroscopy : Measuring spin-relaxation interactions of moderately immobilized spin labels in proteins
- 2004
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 60:2, s. 117-138
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Tidskriftsartikel (refereegranskat)abstract
- Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1) < 0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T-2e, is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-1252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T-2e. We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.
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| 20. |
- Hjerten, Maria, et al.
(författare)
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Renewable enzyme reactors based on beds of artificial gel antibodies.
- 2008
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:6, s. 1188-1191
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach is described for the synthesis of beds for enzyme reactors. The method is based on the use of artificial antibodies in the form of polyacrylamide gel particles with diameters around 0.1-0.3 mm. These gel particles mimic protein antibodies, raised in experimental animals, in the sense that they selectively recognize and adsorb only the protein present during the preparation of the "antibodies". The gel antibodies have several advantages over conventional protein antibodies, which can be taken advantage of in the design of enzyme reactors; for instance, if upon prolonged use the immobilized enzyme loses its activity it can easily be replaced by an active enzyme, which is not possible when the enzyme is immobilized via a conventional protein antibody (a new bed with immobilized protein antibodies must be prepared); and equally or more remarkable: the enzyme can be applied in the form of a non-purified extract since the selectivity of the artificial gel antibodies is so high that they will "fish-out" the enzyme, but no other proteins in the extract. In addition, no preconcentration of the enzyme solution is required prior to the immobilization, since the enzyme is enriched at the top of the column upon the application. These unique properties make enzyme reactors based on artificial gel antibodies very attractive, also in process chromatography. The potential application range of the artificial gel antibodies is enormous since the same method for their synthesis can be used independent of the structure and the size of the "antigen"; for instance, renewable biosensors based on gel antibodies for the selective detection of protein biomarkers, as well as pathogenic viruses, bacteria, and spores (for instance Anthrax) should not be difficult to design.
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| 21. |
- Kalbin, Georgi, et al.
(författare)
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Effects of UV-B in biological and chemical systems : equipment for wavelength dependence determination
- 2005
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 65:1, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- The thinning of the stratospheric ozone layer has prompted a large number of studies of UV-B-induced effects in biological and chemical systems. The wavelength dependency of such effects is of interest from mechanistic, physiological or economic points of view. Here, we describe an apparatus for determining the wavelength dependency of UV-B effects in biological and chemical systems. The apparatus consists of a high intensity UV radiation source and narrow bandpass filters to produce UV radiation in even intervals (between 280 and 360 nm). The usefulness of the equipment is demonstrated in two different systems: 1) Chalcone synthase (CHS) gene is up-regulated by UV-B radiation. Therefore quantitative analysis of the CHS gene expression was chosen in the present investigation for studies of the wavelength dependency of gene expression regulation in plants. Maximum induction of CHS expression was found at 300 nm with a 12-fold induction compared with the control; 2) The wavelength dependency of formation of dioxin-like photoproducts from the brominated flame retardant decabrominated diphenyl ether (DeBDE) is described. This is an example of UV-B-induced conversion of non-toxic species into a number of products of which some may be toxic in the environment. In the UV interval studied, the highest dioxin-like activity was found in the sample irradiated at 330 nm and therefore this wavelength is most important for the mechanism involved in photoconversion of DeBDE.
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| 22. |
- Lammi, Mikko, 1961-, et al.
(författare)
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Autoradiographic quantitation of radiolabeled proteoglycans.
- 1991
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 22:4, s. 301-310
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Tidskriftsartikel (refereegranskat)abstract
- Radiolabeled proteoglycans or glycosaminoglycans were precipitated with the cationic dye safranin O onto a sheet of nitrocellulose filter using a dot-blot apparatus. An autoradiography film was exposed against the nitrocellulose sheet. The developed film and the nitrocellulose sheet were separately digitized in a flat-bed-gray-scale scanner connected to a microcomputer. An image analysis program of the microcomputer was used to quantify the density of the radioactivity dots produced in the film, and the intensity of the dye spots on nitrocellulose. With this procedure, a single sample containing the minimum of about 20 ng uronic acid and 5 dpm of incorporated 35SO4 was quantified for both total glycosaminoglycan content and radioactivity. Unincorporated 35SO4 and low molecular mass radioactivity (e.g. products of glycosaminoglycan degrading enzymes) did not interfere since they were quantitatively washed through the membrane before the assay.
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| 23. |
- Lundqvist, Helen, et al.
(författare)
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Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis.
- 2008
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:6, s. 1059-1065
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Tidskriftsartikel (refereegranskat)abstract
- The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria-neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.
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| 24. |
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| 25. |
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| 26. |
- Masarova, Jana, et al.
(författare)
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Novel peptide surface for reversible immobilization of concanavalin A
- 2004
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 60:2, s. 163-170
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Tidskriftsartikel (refereegranskat)abstract
- Concanavalin A (Con A) was spontaneously adsorbed on polymyxin B surface. This peptide-lectin interaction was strong, KD=1.9×10 -10, based predominantly on creation of hydrophobic bonds, and was completely reversible. Concanavalin A on polymyxin B (PmB) retained higher binding capacity for yeast mannan, compared with covalently immobilized lectin. Kinetics of mannan-concanavalin A interaction were significantly different in dependence on type of concanavalin A immobilization. © 2004 Elsevier B.V. All rights reserved.
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| 27. |
- Ramser, Kerstin, et al.
(författare)
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A combined micro-resonance Raman and absorption set-up enabling in vivo studies under varying physiological conditions : the nerve globin in the nerve cord of Aphrodite aculeata
- 2007
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:4, s. 627-633
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Tidskriftsartikel (refereegranskat)abstract
- We hereby report on the design of a set-up combining micro-resonance Raman and absorption spectroscopy with a microfluidic system. The set-up enabled us to study the nerve globin of Aphrodite aculeata in the functional isolated nerve cord under varying physiological conditions for extended periods of time. The oxygenation cycle of the organism was triggered by utilizing the microfluidic system that allowed for a fast switch between aerobic and anaerobic conditions. The nerve globin was found to very easily shift from a penta-coordinated high spin ferrous form to the oxy state upon a change from anaerobic to aerobic conditions. The observed fast reaction to varying O2 concentrations supports an oxygen-carrying and/or -storing function of the nerve globin. In addition, by combining resonance Raman and absorption spectroscopy, the physiological response could be distinguished from light-induced effects.
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| 28. |
- Rezeli, Melinda, et al.
(författare)
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A new approach for on-line enrichment in electrophoresis of dilute protein solutions.
- 2008
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:6, s. 1098-1103
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Tidskriftsartikel (refereegranskat)abstract
- A method is described for on-line enrichment/zone sharpening of a sample of negatively charged proteins (an analogous method for cationic proteins can be designed). The sample is applied on the top of a 5-mm thick layer of a neutral polyacrylamide gel which rests on another 5-mm thick, large-pore polyacrylamide gel which contains positively charged groups. The latter gel layer is attached to the neutral gel column, used for the electrophoretic separation of the proteins. When a voltage is applied the proteins start migrating and become electrostatically adsorbed at the top of the charged, large-pore gel layer (pH 5.4). With the upper electrode vessel filled with a buffer of a pH higher (pH 7.7) than that employed in the enrichment step and with a voltage between the electrodes, these enriched proteins are released (because the enrichment gel is non-charged at pH 7.7) with zone sharpening and migrate into the 5-cm long column (i.d. 5 mm) of a neutral, large-pore polyacrylamide gel for electrophoretic analysis. Upon the electrophoretic migration from the enrichment gel into the separation gel a second zone sharpening may occur, if the increase in pH from 5.4 to 7.7 in the separation gel is not close to momentary. By employing colored test proteins the efficiency of the enrichment step is visually illustrated by a picture. The principle of the concentration method described has been employed also in chromatographic experiments and can with appropriate modifications also be used in other electrophoretic methods, such as capillary electrophoresis.
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| 29. |
- Råvik, Mattias, et al.
(författare)
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A method for microbial cell surface fingerprinting based on surface plasmon resonance
- 2007
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Ingår i: Journal of Biochemical and Biophysical Methods. - : Elsevier BV. - 0165-022X .- 1872-857X. ; 70:4, s. 595-604
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Tidskriftsartikel (refereegranskat)abstract
- A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).
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| 30. |
- Råvik, M, et al.
(författare)
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A method for microbial cell surface fingerprinting based on surface plasmon resonance
- 2007
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Ingår i: Journal of Biochemical and Biophysical Methods. - 0165-022X .- 1872-857X. ; 70:4, s. 595-604
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Tidskriftsartikel (refereegranskat)abstract
- A method for cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Esherichia coli mutants have been used as model cells, which were cultivated in shake flasks. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences between some of the isolates were observed. Comparative measurements of physical property data are also included. A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA)
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| 31. |
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