Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Synthetic ampholytes for the isoelectric focusing of proteins

Products of the copolymerization of acrylic acid with oligoethylene oligoamines were used to establish natural pH gradients for the isoelectric fractionation of protein mixtures by polyacrylamide gel electrophoresis. The unfractionated synthesized ampholytes were compared with the commercially available ampholytes with respect to the number of ampholyte components, the pH gradient and the resolution of protein mixtures. The performance of the ampholytes prepared employing the more highly substituted ethylene amines was found to be comparable to that of the commercial ampholytes over the pH range 4 to 8.     

Isoelectric focusing of oat root membranes

The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad-range (pH 3-10) or narrow-range (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient. Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures.     

Incorporation of Labeled Precursors into the RNA and Proteins of Lactic Dehydrogenase Virus

Lactic dehydrogenase virus was grown in primary mouse embryo cells and labeled with (3)H-uridine and (3)H-amino acids. Concentrated and purified virus was banded by isopycnic centrifugation in sucrose gradients, and infectivity and radioactivity were found to correspond at a density of 1.17 g/cm(3). The extracted viral RNA was resolved by electrophoresis in polyacrylamide-agarose mixed gels, and the mol wt was estimated to be 6.0 x 10(6).     

Characterization of Aleutian disease virus as a parvovirus.

We characterized a strain of Aleutian disease virus adapted to growth in Crandall feline kidney cells at 31.8 degrees C. When purified from infected cells, Aleutian disease virus had a density in CsCl of 1.42 to 1.44 g/ml and was 24 to 26 nm in diameter. [3H]thymidine could be incorporated into the viral genome, and the viral DNA was then studied. In alkaline sucrose gradients, Aleutian disease virus DNA was a single species that cosedimented at 15.5S with single-stranded DNA from adeno-associated virus. When the DNA was analyzed on neutral sucrose gradients, a single species was again observed, which sedimented at 21S and was clearly distinct from 16S duplex adeno-associated virus DNA. A similar result was obtained even after incubation under annealing conditions, implying that the bulk of Aleutian disease virus virions contained a single non-complementary strand with a molecular weight of about 1.4 X 10(6). In addition, two major virus-associated polypeptides with molecular weights of 89,100 and 77,600 were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virus purified from infected cultures labeled with [35S]methionine. These data suggest that Aleutian disease virus is a nondefective parvovirus.     

A semiautomated system for the production and analysis of sucrose density gradients

A semiautomated system permitting considerable accuracy, speed and reproducibility in the making and fractionation of sucrose density gradients is described. The system consists of a modified Beckman gradient forming device which makes six gradients simultaneously and delivers them into six 12.5 ml polyallomer centrifuge tubes in such a manner that new material is continuously added to the meniscus of the gradient. The gradients are fractionated three at a time and up to 100 fractions per gradient can be collected automatically directly into scintillation vials with a choice of drop counting or time mode with rinse and automatic addition of scintillation fluid to each vial. The system can process up to six gradients per hour but centrifugation time is usually the limiting factor. With neutral sucrose gradients, sharp, reproducible, monodisperse peaks containing up to 100% of the gradient radioactivity are usually obtained but a smaller monodisperse peak containing as little as 3.5% of the gradient radioactivity can be detected under conditions where some pairs of molecules might tangle or dimerize. The resolution and reproducibility of this system when used with neutral sucrose gradients is at least the equal if not superior to that commonly claimed for alkaline sucrose gradients.     

Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.     

Effect of sucrose gradient composition on resolution of RNA species

Resolution of RNA samples on sucrose gradients was strikingly dependent upon the sucrose concentration in the gradients. Linear 10–70% gradients separated peaks which were barely discernible as shoulders in 10–20% gradients. The resolution obtained in steep sucrose gradients was comparable to that observed upon electrophoresis in acrylamide/agarose gels.     

A simple technique for the preparation and storage of sucrose gradients

A method for preparing multiple sucrose gradients by quickly freezing layers of sucrose has been developed. These gradients may be stored in the freezer indefinitely, and thawed from 8 to 24 h at 4 degrees C before use. The middle region of the resulting sucrose gradients was linear. Thawing time and centrifugation had little effect on the shape of the gradient. The method is applicable for both small- and large-volume centrifuge tubes. Gradients prepared in the same batch were nearly identical.     

Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with [3H]thymidine and 14C-amino acids. Infected cells or nuclei prepared from them were extracted with Triton X-100 and NaCl, utilizing a method recently described, and the low-speed supernatant (extract) was partially purified by sedimentation on sucrose gradients. A nucleoprotein complex which sedimented as a wide peak around 200S was identified. The nucleoprotein complex contained viral DNA, which banded at the expected density in CsCl isopycnic gradients and was intact after measurements taken on electron microscopic photographic enlargements. The autoradiographic pattern of 14C-labeled proteins after electrophoresis showed that only a few of the virus-specific polypeptides were present in the nucleoprotein complexes, in particular, VP5, VP12, VP15.2, VP19, and VP24. Cellular histones were absent. The extracts and the nucleoprotein complexes were centrifuged to equilibrium in metrizamide density gradients without prefixation. Electron microscopic direct visualization of the nucleoprotein complexes after sucrose or metrizamide purification revealed that the proteins were preferentially associated with one end of the DNA molecule and formed large irregular terminal thickenings or capsid-like transparent shells enclosing polyglobular cores. No nucleosomes were observed on herpes simplex virus nucleoprotein complexes. The same type of complex was detected after phosphonoacetic acid addition, and grossly altered nucleocapsids were formed.     

Selection of chemical spacers to improve isoelectric focusing resolving power: implications for use in two-dimensional electrophoresis

Presented here is a straightforward and inexpensive method for expanding isoelectric focusing pH gradients relative to the gradients that are formed by commercially available narrow range ampholytes. This method requires no special equipment or techniques and is applicable to isoelectric focusing in acrylamide gels, in Sephadex, and in agarose. The utility of separators in improving the resolving power of two-dimensional polyacrylamide gel electrophoresis is demonstrated using proteins from the exocytotic trichocyst organelle of Paramecium tetraurelia. The mode of action of separators is briefly described.     

Mobility measurements by photometric analysis of zone electrophoresis in a sucrose gradient column

A density gradient zone electrophoresis apparatus has been designed for measuring electrophoretic mobilities on small amounts (about 10 μg) of viruses or other ultraviolet-absorbing materials. Dialysis membranes separate the gradient column from electrode chambers and eliminate the need to maintain hydrostatic equilibrium between the buffer chambers. The virus zone was located by pumping the gradient column through an ultraviolet flow cell, a procedure which did not disturb the virus zone. The apparatus was tested by measuring the mobilities of tobacco mosaic and brome mosaic viruses in different buffers, in sucrose, glucose, and glycerol gradients, at 0° and 15°, and during ascending and descending electrophoresis.     

Iodination of hepatitis A virus reveals a fourth structural polypeptide.

Hepatitis A virus present in the feces of two patients with naturally acquired hepatitis A was purified, radiolabeled with 125I, and analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the three structural polypeptides previously reported, a fourth polypeptide with a molecular weight of 14,000 was detected and shown to be a component of hepatitis A virus by immune precipitation techniques. Intact virions were also shown to sediment at 160S on sucrose gradients. These findings are consistent with hepatitis A virus being an enterovirus within the family Picornaviridae.     

Properties of feline leukemia virus. III. Analysis of the RNA.

The kinetics of virus labeling was used to study the maturation of viral RNA in the Rickard strain of feline leukemia virus. Viral RNA labeled over differing intervals was characterized by gel electrophoresis and velocity sedimentation in sucrose gradients made up in aqueous buffer and 99% dimethyl sulfoxide. Labeled virus was found within 30 min after adding radioactive uridine to the cells and production of labeled virus reached a maximum at 4 to 5 h after pulse labeling. Native RNA from feline leukemia virus resolved into three size classes when analyzed by electrophoresis on 2.0% polyacrylamide-0.5% agarose gels: a 6.2 x 10(6) to 7.1 x 10(6) mol wt (50 to 60S) class, an 8.7 x 10(4) mol wt (approximately 8S) class, and a 2.5 x 10(4) mol wt (4 to 5S) class. From two experiments during which RNA degradation appeared minimal, these made up to 57 to 76%, 2 to 5%, and 6 to 12%, respectively, of the total RNA. The 8S RNA in feline leukemia virus has not previously been reported. The 50 to 60S RNA from virus harvested after 4 h of labeling electrophoretically migrated faster and sedimented more slowly in sucrose gradients than did the same RNA species harvested after 20 h of labeling. This argues for an intravirion modification of the high-molecular-weight RNA. The large subunits of denatured viral RNA from both 4- and 20-h labeled-viral RNA electrophoretically migrated with an estimated molecular weight of 3.2 x 10(6) but sedimented with 28S ribosomal RNA (1.8 X 10(6) mol wt) when analyzed by velocity sedimentation through 99% dimethyl sulfoxide.     

A study of the heterogeneous 37s ribonucleic acid induced by foot-and-mouth-disease virus

1. The 37s RNA induced in baby-hamster kidney cells by infection with foot-and-mouth-disease virus was examined on sucrose gradients and by filtration through Sepharose 4B. 2. The RNA sedimented faster (37s) and as a broader band than the 35s RNA from purified virus. 3. Treatment with deoxyribonuclease, Pronase or amylase did not alter the sedimentation profile of the 37s RNA. 4. Treatment of individual fractions of the RNA with phenol, dimethyl sulphoxide or methylCellosolve did not decrease the sedimentation rate of the faster-sedimenting molecules. 5. Sedimentation in sucrose gradients of different ionic strengths or containing EDTA had no effect on the heterogeneous nature of the profile. 6. On filtration through Sepharose 4B columns, the 37s virus-induced RNA was eluted before viral RNA. 7. Only 20% of the rapidly sedimenting RNA was incorporated into complete virus particles.     

Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.     

Interaction of urokinase with alpha2-macroglobulin investigated by isoelectric focusing. Evidence for non-specific dissociable binding.

The binding of urokinase to human alpha2M (alpha2-macroglobulin) was investigated in comparison with the formation of the equimolar trypsin-alpha2M complex. Experiments were performed by molecular-sieving on Sephadex G-200, subunit conversion by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis after reduction and isoelectric focusing in linear sucrose gradients with ampholytes pH 3.5-10.0. Urokinase activity was determined with alpha-N-acetyl-L-lysine methyl ester and by activation of plasminogen on unheated fibrin plates. alpha2M was determined by single radial immunodiffusion. alpha2M was capable of binding some urokinase by a non-specific type of attachment that could be disrupted by isoelectric focusing but not by gel filtration. The pI of the undissociated trypsin-alpha2M complex was 6.0, and differed from that of the pure alpha2M (5.2-5.4). Likewise the pI of the immunoreactive alpha2M was 5.2 after exposure to urokinase, whereas the dissociated urokinase focused at pI 10.2. This indicated lack of true inhibitor-complex formation, which was also sustained by total absence of subunit conversion. The results are in agreement with our previous findings with pancreatic and urinary kallikreins.