High-voltage Isoelectric Focusing Research Articles

Investigation of CSF proteins using high voltage isoelectric focusing in ultrathin gels and enzyme amplified immuno sandwich assay.

Investigation of CSF proteins using high voltage isoelectric focusing in ultrathin gels and enzyme amplified immuno sandwich assay.

Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High-voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, kappa-chain, lambda-chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti-serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously.

Development of a thermoelectric cooling apparatus for high-voltage isoelectric focusing on a cellulose acetate membrane.

To develop an isoelectric focusing apparatus using a cellulose acetate membrane (Separax EF), we have designed a thermoelectric cooling isoelectric apparatus. This apparatus has two characteristics. Firstly, the cooling system was switched to a thermoelectric cooling system from an ice-cooling system. Secondly, the chamber lid of the electrophoretic apparatus was also devised so that samples could be applied without opening the chamber lid. With this apparatus we could perform the isoelectric focusing without worrying about room temperature and humidity in the laboratory. Applying 2000 V for an extra 5 min with our module cooling system, we achieved a much higher degree of resolution with three sheets of cellulose acetate membrane (Separax EF) overlaid for simultaneous electrophoresis. Thus, three types of information could be obtained from only one electrophoretic procedure.

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.

高圧セルロースアセテート膜等電点電気泳動法による蛋白，糖蛋白，特定蛋白同時３分析法

高圧セルロースアセテート膜等電点電気泳動法による蛋白，糖蛋白，特定蛋白同時３分析法

Purity of Glycera dibranchiata monomer hemoglobin components III and IV determined by isoelectric focusing

1. The Glycera dibranchiata monomer hemoglobin components III and IV display behavior upon high voltage isoelectric focusing which is similar, but not identical to the behavior demonstrated by monomer hemoglobin component II (Constantinidis and Satterlee (1987). Biochemistry 26, 7779-7786). 2. Both components III and IV show multiple line behavior and formation of significant amounts of apoprotein when solutions of each holoprotein are focused on polyacrylamide gels. 3. The apoprotein of each component focuses as a single line, indicating that this is the most unambiguous estimate of purity for these proteins. 4. The purity of the component III and IV preparations can be estimated to be at least 95%.

高圧セルロース・アセテート膜等電点電気泳動法の至適泳動条件に関する基礎的研究

The apparatus, procedure and optimumized electrophoretic conditions for high-voltage isoelectric focusing on cellulose acetate membrane were reported previously. This report focuses on the steps taken to derive at the optimum conditions specified. Temperature of the membrane was maintained below 2°C even at 1500V by cooling with ice water. The membrane was kept moist by the addition of 10% (W/V) sucrose to the carrier-ampholyte solution. Pre-focusing at 500V for 30 minutes was run to obtain a pH gradient on the membrane. 30% (W/V) sucrose was added to the anode solution to prevent drying of the anode wick. The optimum conditions allowed a linear pH gradient between pH 3.5 to 8.5 to be obtained and consequently sharp protein fractionation patterns.

高圧セルロースアセテート膜等電点電気泳動法

高圧セルロースアセテート膜等電点電気泳動法

Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin.

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.

High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF.

Reference intervals for haemoglobins estimated by isoelectric focusing

By means of high-voltage isoelectric focusing in polyacrylamid gel, reference intervals of haemoglobins were determined in erythrocytes from adults, pregnant women, 4-day-old infants and umbilical cord vessel blood.

High-voltage isoelectric focusing with pharmalyte: Field strength and temperature distribution, zone sharpening, isoelectric spectra, and p I determinations

In isoelectric focusing it is theoretically advantageous to use the highest voltages possible. Limitations are due to uneven heat evolution because of uneven conductivity of the individual carrier ampholyte molecules and/or insufficient cooling. The availability of a new carrier ampholyte. Pharmalyte, and a new flatbed apparatus with improved properties has made it possible to perform experiments at very high voltages. The results are discussed in terms of zone sharpness, resolution, and temperature profile. A method is proposed for accurate p I determinations, considering the temperature dependence of p I and the actual temperature in the gel.