A nondenaturing vertical isoelectric focusing polyacrylamide slab gel system suitable for silver staining and electrophoretic blotting

We have devised a nondenaturing vertical isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE) system which is amenable to silver staining and electroblotting. Apart from being accessible, inexpensive, and simple to use, this new methodology overcomes problems inherent in current IEF methods, for example, pH gradient drift, nonuniform cooling, restricted sample volume, and inability to perform electroblotting. Two photopolymerization gel formulas were derived: a 5% acrylamide formula using bisacrylamide (Bis) as the crosslinker and a 6% acrylamide formula using diallyltartdiamide (DATD) as the crosslinker. The 5% acrylamide Bis gel gave excellent resolution and separation of proteins whereas the 6% acrylamide DATD gel expanded slightly during silver staining, resulting in mild band distortions. At least 80 ng of protein per band could be detected by the silver staining protocol devised. Both the DATD and the Bis gels were suitable for electroblot transfer. Parameters to ensure the optimum conditions for reproducible, high resolution vertical IEF-PAGE are described. IEF-PAGE silver staining and electroblotting procedures and silver staining of the nitrocellulose electroblot procedures are also described. The advantages of this methodology over previously published methods are discussed.     

Anti-estradiol antibodies: isoelectric focusing in polyacrylamide gel

Conditions for isoelectric focusing in polyacrylamide gel of IgG globulins have been refined. Using undenatured antibodies against estradiol, preliminary information about structural and functional properties of antibodies was obtained.     

Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting

Conditions have been established for one-dimensional isoelectric focusing using vertical slab gel electrophoresis, followed by immunoblotting, for the measurement of the phosphorylation state of proteins. The method provides a less time-consuming alternative to two-dimensional gel electrophoresis combined with radiolabeling or immunoblotting. The main advantage of the method is that many samples can be analyzed simultaneously. The technique is applied here to the study of a mammalian initiation factor for protein synthesis, eukaryotic initiation factor 2 (eIF-2). The method allows good separation and quantitation of the different phosphorylated forms of the alpha subunit of eIF-2, when used to analyze either purified eIF-2 or eIF-2 contained in complex mixtures. The method is shown to be well adapted to the measurement of rapid phosphorylation/dephosphorylation kinetics in cell extracts, as well as the measurement of the phosphorylation state of eIF-2 in cultured cells. In addition, the method is shown to confirm the existence of a second phosphorylation site on eIF-2. Although eIF-2 has been used for this demonstration of the efficacy of the method, the technique is applicable to a study of the regulation of covalent modification of any polypeptide for which antibodies are available.     

Preparative immobiline isoelectric focusing of plasma apolipoproteins on vertical slab gels

An effective preparative isoelectric focusing method has been developed using the LKB Immobiline system in a vertical slab gel apparatus. Advantages of this procedure are ease of sample application, excellent resolution, and the direct visualization of focused bands. Narrow pH gradients have been used to separate apolipoprotein E3 isoforms (pH gradient 4.9-5.9) and to resolve the apolipoprotein C mixture (pH gradient 4.0-5.0). Recoveries ranged from 40 to 70%. The method should be valuable for protein and isoform purification.     

Further resolution of adult chick hemoglobins by isoelectric focusing in polyacrylamide gel.

Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2(HbD2). Their pI values are 7.53 +/- 0.02, 7.37 +/- 0.02, 6.92 +/- 0.04 and 6.72 +/- 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 ar identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weight and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.     

Flat bed gel isoelectric focusing of neuraminidases.

Technical details and preliminary results are described for a new flat bed gel isoelectric focusing method to study charge heterogeneity in neuraminidases. The method combines simplicity and high resolving power with the capacity to analyse multiple samples in a single experiment. Isoelectric points are obtained which agree favourably with those gained by other methods.     

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.     

Purification, specificity, and hypervariable region sequence of anti-pneumococcal polysaccharide antibodies elicited in a single rabbit.

Four homogeneous antibodies to type VIII pneumococcal polysaccharide (S8) were isolated from the serum of a single rabbit (3322) by affinity chromatography on an S8 immunoadsoebent by utilizing gradient elution with cellobiose and NaCl. The binding properties of these antibodies were determined by a radioimmunoassay with 125I-bovine gamma-globulin-S8. Cellobiose (a disaccharide unit of S8) was the immunodominant group of each of the four antibodies, but each antibody bound to this disaccharide with different relative affinities. The amino acid sequences (positions 0-40) of three of the four antibody light chains were each different both in framework and first hypervariable region sequences. The fourth antibody light chain has a blocked amino terminus. These findings indicate that antibodies elicited by a relatively simple antigen and examined at one time during the course of immunization in a single rabbit may exhibit common specificities for an oligosaccharide determinant, yet have different binding affinities for that determinant as well as different primary structures in the complementarity (hypervariable) regions and framework regions.     

Subsetting of acetylcholine receptor-reactive antibodies by preparative isoelectric focusing.

The antibodies produced against most foreign antigens are composed of a family of immunoglobulins, a family composed of members that are of a number that often reflects the size/complexity of the molecule that stimulates their production. In other words, such responses involve the activation of a "polyclonal" B lymphocyte population. The antibody products of the B cells, although all capable of binding the original antigen, bind at various immunogenic sites (epitopes) on that antigen. Such differences in antigen-binding fine specificity is determined by amino acid residues in the antibody variable region domains found associated with the antigen combining site and tend to have a complimentary biochemistry with the molecule for which they are intended to interact. Furthermore, in addition to amino acid differences that dictate the isotypes and allotypes of antibody molecules, differences in the amino acids that compose the variable regions can produce differences in net charge of particular antibody molecules; thus, families of polyclonal antibodies, all reactive with the same antigen but with different fine specificities, can be separated and, as shown below, purified based on their isoelectric points by preparative isoelectric focusing (pIEF).     

Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis.

A modification of P. H. O'Farrell's (1975, J. Biol. Chem.259, 4007–4021) two-dimensional gel electrophoresis is described. After isoelectric focusing, the cylindrical gels were fixed and stained with Coomassie brilliant blue R before sodium dodecyl sulfate-slab gel electrophoresis in the second dimension. The modification does not alter the protein patterns obtained, but provides sharper spots. In addition, bands are made visible before separation in the second dimension. Moreover, the modification helps to reduce the amount of ampholine in the dye front during electrophoresis.     

A versatile apparatus for polyacrylamide and agarose gel electrophoresis in Plexiglas slab gel molds

A simple vertical slab gel electrophoresis apparatus for analytical, preparative, and two-dimensional electrophoresis is described. The use of permanently sealed Plexiglas acrylic plastic slab gel molds which need to be sealed only at the bottom during gel formation, rather than the glass plate sandwich used in most previous designs, virtually eliminates leakage during gel formation and, in addition, permits the continuous monitoring with ultraviolet light of proteins and nucleic acids labeled with fluorescent dyes during electrophoresis. Results obtainable with this apparatus are equivalent to those achieved in other apparati which are more expensive to fabricate or purchase.     

Composition of an isoelectric focusing gel yielding a broad pH gradient.

An isoelectric focusing gel system is described which produces a pH gradient spanning the range 4–9. When chick brain mitochondrial polypeptides were focused on such a gel, extra polypeptide spots were observed in the basic region which were not seen in a gel prepared by conventional methods.     

A new convenient column for gel electrophoresis,isoelectric focusing,and zonal electrophoresis

A simple preparative electrophoresis column that can be utilized for gel and zonal electrophoresis and isoelectric focusing has been constructed.     

A new isoelectric focusing gel for two-dimensional electrophoresis constructed in microporous hollow fiber membranes

We describe the preparation of IEF tube gels inside a nonwetting microporous plastic tubing. The gel in the tube need not be extruded after the first dimension separation. Instead, the porous structure of the tubes is made wettable, and the proteins are electrophoresed "through-the-wall" into the second dimension PAGE gel. Commercial ampholytes and reagents are suitable for the procedure. A useful p/ range of 4.5-9.5 can be obtained when p/ 3-10 ampholyte mixtures are used. Because of the high surface area of the porous material, precautions must be exercised to reduce oxygen inhibition during polymerization and dehydration of the gel during storage and use. A sheath device is described that satisfies these requirements. The plastic tubes can be disposed of by incineration and pose no biohazard.     

Purification of beta2-microglobulin in by gel filtration and isoelectric focusing.

Urine from persons with elevated excretion of β₂-microglobulin was concentrated and the proteins were separated by molecular sieve chromatography and isoelectric focusing in a granulated gel. A preparation of high purity was obtained.