Rapid and sensitive detection of recombinant soluble proteins in the supernatant of transfected mammalian cells

Cloning and expression of recombinant soluble proteins could be quite a difficult task, especially when it comes to reliably detect minute amounts of the soluble protein in the supernatant of transfected mammalian cells. Timing and sensitivity are of the essence in order to optimise the benefits/costs balance and to decide which clones to grow further and which ones to discard. Here we propose a modified inhibition assay. The key feature of this approach is the development of a sensitive and quantitative test to detect the presence of the recombinant soluble protein by exploiting its ability to compete with the binding of a specific monoclonal antibody to a target cell. The described procedure is a sensitive, efficient, dependable and low cost method.     

A fast and sensitive method for detecting specific viral RNA in mammalian cells.

A quick and sensitive method to quantitate viral RNA synthesis has been developed. Utilizing glutaraldehyde to fix infected cells onto nitrocellulose paper, viral RNA can be probed directly in situ. Viral message can be detected from as few as 10(4) infected cells. This technique can be used to study viral gene expression and can be adapted to screen the activity of antiviral agents such as interferon.     

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.     

One-step split GFP staining for sensitive protein detection and localization in mammalian cells

Although epitope tags are useful to detect intracellular proteins and follow their localization with antibodies, background and nonspecific staining often remain problematic. We describe a simple assay based on the split GFP complementation system. Proteins tagged with the 15-amino acid GFP 11 fragment are detected with a solution of the recombinant nonfluorescent complementary GFP 1-10 fragment to reconstitute a fluorescent GFP. In contrast to antibody-based staining methods, this one-step assay presents high specificity and very low background of fluorescence, thus conferring higher signal-to-noise ratios. We demonstrate that this new application of the split GFP tagging system facilitates detection of proteins displaying various subcellular localizations using flow cytometry and microscopy analysis.     

New, small circular DNA in transfected mammalian cells.

Circular DNA isolated by the Hirt procedure from transfected mammalian cells was examined by electron microscopy. Typically, the number of small (1- to 5-kilobase) DNA circles increased about fivefold even though DNA of larger size classes (5 to 15 kilobases) has been transferred. In one case, where extensive rearrangement of the transferred DNA was observed, the rearrangement products were cloned and analyzed. In most cases, however, no rearrangement could be detected, but the amount of small circular DNA was still increased. This effect was seen with two transfection procedures (erythrocyte ghost fusion and calcium phosphate precipitation) and with various combinations of transfecting DNA and recipient cell type. The origin of the new small circular DNA is discussed.     

A preparative method for obtaining enucleated mammalian cells.

Analysis of the 2D gel electrophoretic pattern of ribosomal proteins from both the small and large subunit of rat liver were made at various times following partial hepatectomy. No changes were observed in the electrophoretic mobility of proteins from the 60S subunit during periods of 2 hr. to 72 hr. of liver regeneration. Changes were observed, however, in two proteins of the 40S subunit a short time after partial hepatectomy. Protein S₆ disappeared from its normal position and a new spot appeared as a more negative form as early as 2 hr. post regeneration. This modification persisted for at least 18 hr. At 72 hr., S₆ returned to its normal position. Protein S₂, on the other hand, underwent a different pattern of change during the early stages of liver regeneration. S₂ was observed to migrate as 2 spots at 2 hr. after partial hepatectomy and this pattern was preserved at the 4 hr. period. At 8 hr., the pattern was further modified to 2 spots which was distinct from the earlier change. This pattern was similar at 12 hr. At 18 hr. only the normal S₂ protein was observed. No further change in S₂ migration was observed at the 72 hr. period of liver regeneration.     

Effects of methotrexate on transfected DNA stability in mammalian cells.

The chromosomal locations, amounts, and level of expression of transfected, amplified c-myc and dihydrofolate reductase sequences were measured in cells cultured in the presence and absence of methotrexate. These studies show that the location and amount of transfected sequences, as well as the level of expression, were more variable when the cells were cultured in methotrexate.     

A method for enucleating cultured mammalian cells

A method is described for enucleating cells which normally could not be enucleated due to their poor adhesion to the growth surface. The technique consists of linking ConA to the surface and then applying the cells. This results in cell adhesion firm enough to withstand the centrifugal forces necessary to enucleate. The method has been applied to fibroblastic, epithelioid and lymphoid cell lines.     

A radioimmunocytological quantitative method for the rapid detection of ras oncogene p21 protein in mammalian cells

In order to provide a sensitive and quantitative detection method of ras p21 at the cytological level, the monoclonal antibody Y 13 259 and iodinated protein A were used to locate theras protein in various mammalian cell lines. The subsequent autoradiograph can be analysed by a computer-assisted system which showed in these reported experiments that the relative levels of p21 detected in these cells corresponded to results obtained earlier using conventional biochemical methods.To whom correspondence should be addressed.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

A simple and sensitive staining method for the detection of cellulase isozymes in polyacrylamide gels.

A simple and rapid staining procedure is described for qualitative and quantitative determination of the activity of plant (Citrus sinensis (L.) Osbeck cv. Shamouti) and fungal (Trichodermata viride) cellulases in polyacrylamide gels. The method is based on the incorporation of carboxymethyl cellulose, a cellulase substrate, into the gels. After electrophoresis of crude extracts the gels are incubated in sodium-potassium phosphate buffer for the cellulase reaction which is stopped at the desired time by acidification of the gels in 60% sulfuric acid. The gels are then exposed to 2.0% KI + 0.2% I₂. No color develops in areas containing cellulase activity. The experimental procedure is described, and its different aspects are discussed.     

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.