Fluorescent labeling of proteins. A new methodology

Weigele, Manfred; Bernardo, Silvano De; Leimgruber, W.; Cleeland, Roy; Grünberg, E.

doi:10.1016/0006-291x(73)90779-1

Cited by 76 publications

(30 citation statements)

References 9 publications

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“…Furthermore, the emission spectrum of proteins labeled with MDPF does not overlap with the background emission (maximum at 405 nm) produced by the TCPO-H,O, reaction in absence of fluorophors [27, 281. An additional advantage of MDPF is that it is intrinsically nonfluorescent but forms fluorescent derivatives upon reaction with primary amino groups of proteins, while excess reagent is hydrolyzed and forms a nonfluorescent product [21]. We have observed an equivalent behavior in the chemiluminescent system analyzed in this paper.…”

Section: Compatibility Of Different Fluorescent Labels With the Tcpo-mentioning

confidence: 79%

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing

Alba¹,

Daban²

1997

Electrophoresis

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We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.

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Section: Compatibility Of Different Fluorescent Labels With the Tcpo-mentioning

confidence: 79%

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing

Alba¹,

Daban²

1997

Electrophoresis

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“…Forty-five microliters of the dissolved immunoprecipitates were mixed with [4][5][6][7][8] Mg of the appropriate fluorescent (MDPF I)-conjugated standard polypeptide (23,24) and the samples were subjected to electrophoresis on polyacrylamide gels in the presence of NaDodSO4 (0.1%) at 3 mA/tube for S3 hr (18). The reaction of the standard proteins with MDPF I allowed direct visualization of the protein bands both during and after disc-gel electrophoresis by using long-wavelength (352 nm) UV illumination.…”

Section: Uniformlymentioning

confidence: 99%

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

Zarucki-Schulz¹,

Jerez²,

Goldberg³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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The in vitro synthesis of elongation factor (EF)-Tu (tufB), the #I' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage Xrifdl8, EF-Tu (tufA), EF-G, and the a subunit of RNA polymerase directed by DNA from the transducing phage Xfus3 has been investigateydin a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the a and Y' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates gB-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the ,' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.DNA-directed in vitro protein-synthesizing systems afford a valuable tool with which to study the regulation of gene expression (for review, see ref. 1). Because of the complexity of the system, these in vitro studies have used relatively crude extracts to obtain protein synthesis. However, a great deal of new information might be obtained if gene expression could be achieved in a more defined system. Our laboratory has initiated studies on the DNA-directed in vitro synthesis of 13-galactosidase with the hope of obtaining synthesis of this protein in a completely defined system (2-7). It has been possible to obtain significant synthesis of /3-galactosidase in a partially defined system that contained many highly purified factors in addition to several partially purified fractions (7).In conjunction with the studies on the synthesis of 13-galactosidase, experiments were initiated on the expression of the two elongation factor (EF)-Tu genes, tufA and tufB (Fig. 1), carried by the transducing phages Xfus3 and Xrifdl8, respectively (8,9). In addition, quantitative assays have been developed to measure the expression of other genes on these phages, such as ribosomal proteins L10 and L12, EF-G, and the a and 13' subunitst of RNA polymerase (see Fig. 1). The results of these studies using a crude and partially defined system are described here. Bacteriophages Xrifdl8 and Xfus3 were isolated from Escherichia coil H105 (J. B. Kirschbaum, Harvard University) and E. coli N01380 (M. Nomura, University of Wisconsin), respectively. The phages were purified and DNA was extracted as described (10, 11). Preparation, from E. coli Z19iq (provided by G. Zubay, Columbia University), of the ribosomal wash, washed ribosomes, and a supernatant extract (S-200) has been reported (2, 12). The S-200 ...

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“…Proteins were conjugated with MDPF according to the procedure of Weigele et al (22). All conjugates were purified by dialysis in 50,000 molecular weight cut-off tubing (Spectrapor, Fisher) against two changes of 100 vol of 0.02 M phosphate-buffered saline, pH RESULTS Differential Ingestion of Albumin.…”

mentioning

confidence: 99%

Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

Williams¹,

Devenny²,

Bitensky³

1981

Proc. Natl. Acad. Sci. U.S.A.

128

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Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and nonenzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction ofalbumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentration of 6% with respect to total albumin (the level found in "non diabetic" serum), only glycosylated albumin was ingested. At higher concentrations of glycosylated albumin (those found in diabetic serum), both albumin and glycosylated albumin are ingested. Glycosylation of endothelial membrane components results in stimulated ingestion of glycosylated albumin, persistent exclusion of unmodified albumin, and unaltered micropinocytic ingestion of native ferritin. These results indicate that nonenzymatic glycosylation of serum albumin may result in rapid vesicle-mediated extravasation of albumin. Chronic microvascular leakage of glycosylated albumin could contribute to the pathogenesis of diabetic microangiopathy.Molecular "languages" shape the complex interactions that support life processes. Unique symbols, fidelity of transcription, and rules of grammar are features of the genetic code (1). Specificity ofrecognition and informational content are also apparent in the interactions ofhormones with receptors (2), antigens with antibodies (3), and substrates with enzymes (4). In each ofthese examples, accurate recognition of a unique molecular conformation is crucial for the underlying process. Moreover, lapses in the fidelity of recognition can produce catastrophic results for the systems involved.Endothelial micropinocytosis provides a bidirectional large pore conduit for the transendothelial transport of macromolecules. Another form of molecular language is manifest in the interactions of such molecules with the caveolar (plasmalemmal vesicle) membrane components of endothelial cells. The use of this molecular language in the process ofrecognition-dependent endocytosis provides a discrimination function for transendothelial transport. There results a striking heterogeneity in the rates oftransendothelial vesicular transport ofa variety ofserum components (5-8).The reasons for these differences must be sought in specific interactions between each of the components and putative recognition sites in lumenal endothelial membranes. Such sites are situated within or adjacent to the stomata of caveolae and actively regulate the process of adsorptive endocytosis. Serum albumin appears virtually excluded from ingestion by micropinocytic vesicles (5). This exclusion cannot be attributed solely to the molecular dimensions or the net charge of albumin. Ferritin, which is larger and of similar net charge, is readily ingested by endothelial vesicles. The fine molecular details that govern these recognition processes, and thus modula...

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Fluorescent labeling of proteins. A new methodology

Cited by 76 publications

References 9 publications

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

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