Application of schlieren optics to real-time monitoring of protein electrophoresis in crosslinker-free linear polyacrylamide solution

Molecular sieving effect of crosslinker-free linear polyacrylamide solutions could be visualized in a real-time mode using schlieren optics for electrophoresis of proteins. Since linear polymer solutions are going to find application in capillary electrophoresis (Zhu, M., et al., J. Chromatogr. 1989, 480, 311-319), the above approach may be useful for obtaining basic data of electrophoresis of proteins and nucleic acids in such media under conditions free from the predominant wall effect pertinent to the use of a capillary.     

A continuous acetic acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines

A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate-lactic acid system (using 12% acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.     

Determination of protein spots separated by two-dimensional polyacrylamide gel electrophoresis

A simple method for quantitating proteins in the spots on two-dimensional polyacrylamide gel electropherograms is described. The system consists in three steps: (1) O'Farrell's two-dimensional gel electrophoresis of the proteins to be analysed; (2) staining of the gels with Coomassie brilliant blue; and (3) determination of the area and integrated density of the stained spots by the Joyce Loebl Magiscan-1 image analysis system. The method can be used for the determination of proteins in the range 0.5-100 micrograms/cm2; the amount of protein involved in most spots detected by the staining method actually falls within this range. As the minimum spot diameter that can easily be handled by the method is about 2 mm, as much as 30 ng of protein in such a spot can be determined. The method can also be applied to autoradiograms.     

Optimizing soluble protein extraction and two-dimensional polyacrylamide gel electrophoresis quality for extremophile ciliates

An efficient protein extraction methodology is quite important for sample preparation and subsequent 2-D PAGE and MS analysis. Cell lysis is the first step in protein extraction and purification. Many techniques are available for cell disruption, including physical and detergent-based methods. Here, we report on a very fast and efficient detergent-free Tris-based method to extract the soluble fraction proteins of extremophile ciliates, comparing it with a detergent-based protocol. This comparison has been carried out by means of 2-D PAGE and subsequent MALDI-compatible silver staining of protein samples obtained from the intensely pigmented hypersaline ciliate Fabrea salina and the Antarctic hypotrich ciliate Euplotes focardii. Our results indicate that this fast and easy extraction method allows to obtain more clear crude extracts and more spot-abundant polyacrylamide gels.     

Localization of separated protein bands in unstained electrophoresed polyacrylamide gradient slab gel.

Polyacrylamide gel electrophoresis has become the most widely used separation method in biological science. Once electrophoresis is complete the protein bands must be localized prior to excision. A zig-zag gel cutter is described which cuts a strip of gel from the side of a slab gradient gel or a gel of uniform concentration in peaks and valleys. The location of the protein of interest is determined by counting the number of peaks on the stained side strip. The portion of the unstained gel corresponding to the same count (number of valleys) is cut to recover the protein of interest from the main gel for further manipulations.     

Genomically linked cellular protein databases derived from two-dimensional polyacrylamide gel electrophoresis

In its most useful form a cellular protein database should be genomically based, because it is the genome which determines both the total number of proteins a cell can make and the particular ones that will be made under any given condition. Such a database should trace each protein back to its structural gene, and should account for every structural gene of a cell. Recent advances in molecular biology greatly facilitate the construction of such gene-protein databases. The mapping of genes of unidentified proteins resolved from total cell extracts on two-dimensional gels can now be accomplished by largely biochemical methods, without the necessity of isolating mutants or performing genetic crosses. Other techniques permit one to search gels for the product of any newly discovered gene (or open reading frame) suspected of encoding a protein. Consequently, gene-protein indices can be built independently and simultaneously from either direction--deducing the genetic map from the protein pattern, or finding the protein pattern from information encoded in the genome. A database of this sort is being constructed for the bacterium, Escherichia coli. Given the current pace of DNA nucleotide sequencing, the development of total gene-protein indices for a variety of cells can be anticipated in the near future.     

Microsequencing of proteins electrotransferred onto immobilizing matrices from polyacrylamide gel electrophoresis: application to an insoluble protein

Semidry electroblotting is convenient and allows a rapid and efficient protein transfer from one- or two-dimensional polyacrylamide gels onto sequencer stable supports for protein microsequence analysis in a gas-phase sequencer. Using this technique, we determined the amino acid sequences of the basic 7S globulin (Bg), an insoluble protein present in soybean seeds. Based on sequence determination, the cDNA-encoding Bg could be easily cloned and characterized.     

A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.

A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.     

Carbon nanotubes-assisted polyacrylamide gel electrophoresis for enhanced separation of human serum proteins and application in liverish diagnosis

The application of pore-gradient polyacrylamide gel electrophoresis (PG-PAGE) incorporated with carbon nanotube modified by Triton X-100 and carboxylation so as to improve the separation of human serum proteins is reported. The novel PG-PAGE was made by adding water-soluble single-walled carbon nanotubes (CNTs) when preparing the polyacrylamide gel. Significant improvements in separation of complement C3 protein and haptoglobin (Hp) in human serum were achieved. It was estimated that the interactions between the hydrophilic groups on the proteins and the surface of the CNTs result in different adsorption kinetics of complement C3 and Hp subtype on the nanoparticles incorporated in the gel, thus enhancing the separation of the two proteins in serum. This new CNT matrix-assisted PG-PAGE method for enhanced separation of complement C3 and Hp in human serum was successfully applied to distinguish the samples from liverish patients and healthy people.     

The application of two-dimensional polyacrylamide gel electrophoresis to medical microbiology: molecular epidemiology of viruses and bacteria

A variety of molecular methods can be used to identify protein and nucleic acid markers with which to investigate the epidemiology of viruses and bacteria. This paper reviews the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) for studying microbial molecular epidemiology. A small format 2-D PAGE system is described for locating protein markers in group B coxsackie viruses (CVB) and Haemophilus influenzae isolates. Representative isolates of CVB serotypes 2, 4, and 5 were compared by analysing the intracellular proteins present in CVB-infected HEp-2 cells by 2-D PAGE protein gels. Although some of the virus-induced proteins had similar electrophoretic mobilities, the three serotypes could be distinguished from each other on the basis of a major virus-induced protein of molecular weight between 39,000 and 43,000. Protein differences were demonstrated among six serotype 2 CVB (CVB-2) isolates. Four clinical CVB-2 isolates collected over a period of four months had indistinguishable two-dimensional protein profiles. Comparison of the two-dimensional protein profiles of cloned virus stocks prepared from a single clinical CVB isolate demonstrated that it was a heterogeneous virus population. The proteins of nontypable and type-b H. influenzae isolates were compared. Up to 160 proteins, detected by staining with Coomassie Brilliant Blue R, were resolved by 2-D PAGE. Although protein differences between individual bacterial isolates were detected, comparable two-dimensional protein profiles were found for the two groups of H. influenzae isolates. There was no similarity in the two-dimensional protein profiles of H. influenzae and Aeromonas. Potential protein markers were identified that may be useful in long-term studies of H. influenzae epidemiology.     

Report of workshop on cellular protein databases derived from two-dimensional polyacrylamide gel electrophoresis

A workshop entitled Cellular Protein Databases from Two-Dimensional Gel Electrophoresis was held in Atlanta, Georgia, 28 February-1 March 1987. Its purpose was to assess the status of two-dimensional gel electrophoresis of proteins as a research methodology in biological systems, particularly in the generation of cellular protein databases. The workshop participants summarized current studies on a variety of biological systems, both prokaryotic and eukaryotic. Analysis of the progress being made led to the conclusion that electrophoretic techniques, supported by automatic scanning of gel images and computer-assisted processing, analysis and matching of gel images, are now capable of generating databases of great potential value. Factors affecting the reproducibility of protein spot patterns on gels were identified, and the extent to which gel pattern variability causes difficulties in communicating results and in integrating information from different laboratories was assessed. Measures were suggested to help overcome obstacles to the generation of comprehensive cellular protein databases from the electrophoretic resolution of total cellular proteins.     

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis

In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution.