Isoelectric Focusing Of Proteins Research Articles

Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution

A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4–5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes possess mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.

Zonal electrophoresis and isoelectric focusing of proteins and virus particles in density gradients of small volume

Proteins and virus particles were separated by zonal electrophoresis or isoelectric focusing in glass tubes of small volume. The tubes were covered at the bottom with dialysis membranes and sucrose gradients containing either buffer or ampholytes were generated directly into them. When ampholytes were employed, reproducible pH gradients were generated during electrophoresis. After the separations were finished, dense sucrose was pumped into the bottom of each tube and the gradient was fractionated from the top; the recovery of virus was nearly complete.

Isoelectric focusing of proteins in gels

The method of isoelectric focusing in a gel has become widely used in spite of the fact that the first papers on this question were published only four years ago. This is due to the advantages of the method: high sensitivity and resolving power, simplicity and rapidity, and possibility of combination with other methods.

Isoelectric focusing of proteins at the 10 −10 to 10 −9-g level

A microisoelectric focusing method is described which is sensitive at the 10 −10 g level, and is comparable in its pH gradient characteristics to conventional isoelectric focusing methods in acrylamide gels.

Isoelectric focusing of proteins in polyacrylamide gels

An apparatus for isoelectric focusing in gel slab, thin layer or flat bed is described. Furthermore the following points are described: (1) the procedure for making a suitable gel, (2) application of protein in different ways, (3) detection of protein especially by staining, (4) pH measurement, (5) sectioning the gel. Suitable concentrations of acrylamide and carrier ampholytes are discussed. A comparison is made between advantages of isoelectric focusing in gel rod and gel slab. Most arguments are in favour of the latter technique.

Isoelectric focusing of proteins on bactopeptone

Isoelectric focusing of proteins on bactopeptone

Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin.

1. An analytical technique of isoelectric focusing in thin layers of polyacrylamide gel has been used to determine the isoelectric point, pI, of several proteins in the presence and in the absence of concentrated urea. 2. The presence of urea did not greatly affect pI except for bovine plasma albumin, where an increase of approx. 1pH unit was found. 3. Evidence is presented that this change in the pI of bovine plasma albumin is due to the normalization of certain ionizable groups on unfolding of the protein in urea. 4. Evidence is also presented that prolonged exposure of bovine plasma albumin to urea results in intramolecular disulphide interchange and that, on removal of urea, the new patterns of disulphide bonding stabilize abnormal conformations with pI values intermediate between those of the native and denatured states. 5. The studies demonstrate heterogeneity in bovine plasma albumin based on primary-sequence differences. 6. Isoelectric focusing of proteins in urea appears to be useful in the study of various aspects of protein structure.

Isoelectric points and conformation of proteins: I. Effect of urea on the behavior of some proteins in isoelectric focusing

As an attempt to explore a new method for studying conformation of proteins, the isoelectric points of human hemoglobin, bovine insulin, bovine pancreatic ribonuclease, human serum albumin and hog thyroglobulin were examined by isoelectric focusing in the absence and presence of 6 M urea. After appropriate corretion was made for the apparent effect of urea on the measured pH of aqueous urea solutions, no variation in the isoelectric point in the presence of urea was found with hemoglobin and insulin. On the other hand, three other kinds of protein showed a definite increase in the isoelectric point in the presence of 6 M urea. In the case of human serum albumin, a large extent of heterogeneity observed with the native protein was markedly reduced in 6 M urea, whereas the microheterogeneity detected with thyroglobulin did not show such a tendency. A method is proposed to relate the observed shift in isoelectric point caused by urea to the anomalous dissociating groups that might be present in the native molecule and responsible for this phenomenon. The results are discussed on the basis of information hitherto obtained by other procedures. It is concluded that isoelectric focusing is not only useful for the fractionation of protein mixtures and for the determination of isoelectric points of proteins, as currently accepted, but also valuable for conformational studies of some proteins, especially those with known chemical structure.

Gel isoelectric focusing of cerebrospinal fluid proteins: a potential diagnostic tool.

Isoelectric focusing of proteins is now well established as a powerful tool in protein chemistry following its introduction a few years ago (1, 2). Unfortunately, this new technique has not yet found practical day to day use in the·, clinical laboratory. This communication describes a reproducible and easy method for gel isoelectric focusing (3, 4, 5)! It also contains the preliminary' results obtained with cerebrospinal fluid proteins, mostly from multiple sclerosis cases.

Isoelectric Focusing of Proteins in Gel Media

Abstract Separation of proteins according to their isoelectric point can be performed in a pH gradient formed by stationary electrolysis of carrier ampholytes. The pH gradient is stabilized by the use of polyacrylamide, agarose, and Sephadex gels. Separated proteins can be detected by fixation with trichloroacetic acid followed by nonspecific staining, by specific staining, or through immunodiffusion techniques. Isoelectric focusing of proteins in gel media can be carried out in gel columns or on thin-layer plates by using conventional electrophoresis apparatus. Electrofocusing can be followed by electrophoresis in gel media for more complete separation of components. Multiple samples of microgram quantities can be analyzed simultaneously by simple and rapid procedures. These methods have both analytical and preparative applications in protein fractionation work.