Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

scholarly journals Evidence concerning the mechanism of insulin-receptor interaction and the structure of the insulin receptor from biological properties of covalently linked insulin dimers

1983 ◽  
Vol 216 (3) ◽  
pp. 687-694 ◽  
Author(s):  
M A Tatnell ◽  
R H Jones ◽  
K P Willey ◽  
A Schüttler ◽  
D Brandenburg
Keyword(s):  
Insulin Receptor ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Biological Properties ◽  
Receptor Interaction ◽  
Cross Linking ◽  
Receptor Complex ◽  
Biological Responses ◽  
Rat Liver Plasma Membranes ◽  
Covalently Linked

Covalently linked insulin dimers have been prepared by cross-linking two insulin monomers with a flexible suberoyl chain at either the B1 phenylalanine or the B29 lysine residue. Binding potencies of dimers determined by inhibition of binding of 125I-insulin to isolated rat liver plasma membranes or adipocytes were 2.5-7-fold greater than their abilities to stimulate lipogenesis in adipocytes. Rates of liver plasma-membrane-associated degradation of labelled insulin and dimers, measured by gel filtration, were similar at 37 degrees C. Binding and lipogenesis potencies of dimers prepared by substitution of each monomeric half of an asymmetrical dimer with desoctapeptide insulin, an almost inactive derivative, implicated the B1-cross-linked monomeric half as predominantly interacting with the insulin receptor. These results suggest that (1) dimers bind univalently to a bivalent insulin-receptor complex, in which the two individual binding subunits are arranged with anti-parallel symmetry and (2) the mechanism by which insulin binds and initiates its biological responses requires a conformational change within the insulin-receptor complex and/or in the insulin molecule for full biological expression.

Combined Cy3 / Nanogold Conjugates for ImmunocytoChemistry and in Situ Hybridization

1999 ◽  
Vol 5 (S2) ◽  
pp. 478-479
Author(s):  
Richard D. Powell ◽  
Vishwas N. Joshi ◽  
Carol M. R. Halsey ◽  
James F. Hainfeld ◽  
Gerhard W. Hacker ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Gold Cluster ◽  
Gel Filtration ◽  
Single Step ◽  
Cyanine Dye ◽  
Fab Fragments ◽  
Cluster Label ◽  
Rabbit Igg ◽  
Gold Probe

Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab’ immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling. We now describe Fab’ and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3.F(ab’)2 Goat anti-Mouse IgG and F(ab’)2 goat anti-rabbit IgG fragments were reductively cleaved to Fab’ fragments using dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA), which selectively reduce the F(ab’)2 hinge disulfide bonds, with 5 mm EDTA to prevent reoxidation. Fab’ fragments were isolated by gel filtration (coarse gel: GH25, Amicon) then labeled with Monomaleimido- Nanogold® which reacts site-specifically with thiols. Streptavidin was labeled using Mono- Sulfo-NHS-Nanogold® at pH 7.5. Nanogold® conjugates were isolated by gel filtration (Superose-12 column, Pharmacia), then reacted with excess Cy3 monofunctional NHS ester (labeling kit, Amersham Life Sciences) at pH 7.5; dual-labeled conjugates were isolated by gel filtration (Superose-12).

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology

1979 ◽  
Vol 183 (3) ◽  
pp. 615-622 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Sequence Homology ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Terminal Sequence ◽  
Complement Components ◽  
Factor B ◽  
Terminal Residue ◽  
Covalently Linked

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

scholarly journals Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions

1986 ◽  
Vol 234 (1) ◽  
pp. 43-48 ◽  
Author(s):  
E J Bergey ◽  
M J Levine ◽  
M S Reddy ◽  
S D Bradway ◽  
I Al-Hashimi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Gel Filtration ◽  
Cross Linking ◽  
Oral Streptococci ◽  
Bacterial Surface ◽  
Acetylneuraminic Acid ◽  
Streptococcus Sanguis ◽  
Surface Examination ◽  
Inhibition Studies

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.

Site-Specific Cross-Linking of Human and Bovine Hemoglobins Differentially Alters Oxygen Binding and Redox Side Reactions Producing Rhombic Heme and Heme Degradation†

Biochemistry ◽  
2002 ◽  
Vol 41 (23) ◽  
pp. 7407-7415 ◽  
Author(s):  
Enika Nagababu ◽  
Somasundaram Ramasamy ◽  
Joseph Moses Rifkind ◽  
Yiping Jia ◽  
Abdu I. Alayash
Keyword(s):  
Cross Linking ◽  
Side Reactions ◽  
Oxygen Binding ◽  
Site Specific ◽  
Heme Degradation

scholarly journals Alteration of the substrate specificity of human lysozyme by site-specific intermolecular cross-linking

FEBS Letters ◽  
1994 ◽  
Vol 355 (3) ◽  
pp. 271-274 ◽  
Author(s):  
Michiro Muraki ◽  
Yoshifumi Jigami ◽  
Kazuaki Harata
Keyword(s):  
Substrate Specificity ◽  
Cross Linking ◽  
Human Lysozyme ◽  
Site Specific

scholarly journals La antigen recognizes and binds to the 3'-oligouridylate tail of a small RNA.

1984 ◽  
Vol 4 (6) ◽  
pp. 1134-1140 ◽  
Author(s):  
M B Mathews ◽  
A M Francoeur
Keyword(s):  
Rna Polymerase ◽  
Small Rna ◽  
Rna Polymerase Iii ◽  
Cellular Protein ◽  
Cross Linking ◽  
Antigen Binding ◽  
Polymerase Iii ◽  
La Antigen ◽  
Covalently Linked

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.

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