'An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently ... More
Plasticity of B cell receptor internalization upon conditional depletion of clathrin.
AuthorsStoddart A, Jackson AP, Brodsky FM
JournalMol Biol Cell
PubMed ID15716350
'B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized ... More
'Array-based sensors provide an architecture for multianalyte sensing. In this paper, we report a new approach for array fabrication. Sensors are made by immobilizing different reactive chemistries on the surfaces of microspheres. Sensor arrays are prepared by randomly distributing a mixture of microsphere sensors on an optical substrate containing thousands ... More
Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro.
AuthorsSorger PK, Severin FF, Hyman AA
JournalJ Cell Biol
PubMed ID7962081
'Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 ... More
Retention of biologic activity of human epidermal growth factor following conjugation to a blood-brain barrier drug delivery vector via an extended poly(ethylene glycol) linker.
AuthorsDeguchi Y, Kurihara A, Pardridge WM
JournalBioconjug Chem
PubMed ID9893961
'Human brain gliomas overexpress the receptor for epidermal growth factor (EGF), and radiolabeled EGF is a potential peptide radiopharmaceutical for imaging human brain tumors, should this peptide be made transportable through the blood-brain barrier (BBB) in vivo. Peptide drug delivery to the brain may be facilitated by conjugating peptide radiopharmaceuticals ... More
Biotin derivatives of D-Phe-Pro-Arg-CH2Cl for active-site-specific labeling of thrombin and other serine proteinases.
AuthorsAnderson PJ, Bock PE
JournalAnal Biochem
PubMed ID11554721
'Biotin derivatives of peptide chloromethyl ketones have ideal properties for specific labeling of the catalytic sites of serine proteinases but have not been widely used as probes because of the difficulty of synthesis and their instability. To make the reagents more accessible, a simple, economical method was developed for preparation ... More
Dynamic localization of CLIP-170 to microtubule plus ends is coupled to microtubule assembly.
AuthorsDiamantopoulos GS, Perez F, Goodson HV, Batelier G, Melki R, Kreis TE, Rickard JE
JournalJ Cell Biol
PubMed ID9885247
'CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends ... More
Biomolecular recognition on well-characterized beads packed in microfluidic channels.
'We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer ... More
Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.
AuthorsBuranda T, Lopez GP, Keij J, Harris R, Sklar LA
JournalCytometry
PubMed ID10451503
'BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their ... More
Coupling of monoclonal antibodies with biotin.
AuthorsHaugland RP, You WW
JournalMethods Mol Biol
PubMed ID7550685
Coupling of antibodies with biotin.
AuthorsHaugland RP, You WW
JournalMethods Mol Biol
PubMed ID9664374
Preparation of modified tubulins.
AuthorsHyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T
JournalMethods Enzymol
PubMed ID2034137
Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling.
AuthorsHenegariu O, Bray-Ward P, Ward DC
JournalNat Biotechnol
PubMed ID10700155
The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for ... More
N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP.
AuthorsVacca F, D'Ambrosi N, Nestola V, Amadio S, Giustizieri M, Cucchiaroni ML, Tozzi A, Velluz MC, Mercuri NB, Volonté C,
JournalGlycobiology
PubMed ID21186285
N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of ... More
Biotinylation reagents for the study of cell surface proteins.
AuthorsElia G,
JournalProteomics
PubMed ID18763706
The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for derivatization with biotin of a variety of molecules (in particular, proteins) have been introduced, in order to allow their efficient recovery, immobilization and detection with avidin-based reagents. ... More
A method for biotin labeling of biologically active oligogalacturonides using a chemically stable hydrazide linkage.
AuthorsRidley BL, Spiro MD, Glushka J, Albersheim P, Darvill A, Mohnen D
JournalAnal Biochem
PubMed ID9193702
Oligogalacturonides (oligomers of alpha-1,4-D-galacturonic acid) with degrees of polymerization (DP) between 8 and 16 were labeled with biotin using a rapid and simple two-reaction protocol that yields a stable oligogalacturonide derivative. In the first reaction biotin-x-hydrazide was coupled to the anomeric carbon of the reducing galacturonic acid residue by a ... More
Acetylcholinesterase dynamics at the neuromuscular junction of live animals.
AuthorsKrejci E, Martinez-Pena y Valenzuela I, Ameziane R, Akaaboune M
JournalJ Biol Chem
PubMed ID16455662
At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely ... More
Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.
AuthorsHu B, Finsinger D, Peter K, Guttenberg Z, Bärmann M, Kessler H, Escherich A, Moroder L, Böhm J, Baumeister W, Sui SF, Sackmann E
JournalBiochemistry
PubMed ID11015207
We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer ... More
Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins.
c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory ... More
Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.
AuthorsBuranda T, Lopez GP, Simons P, Pastuszyn A, Sklar LA
JournalAnal Biochem
PubMed ID11700971
Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, ... More
Biotinylated epidermal growth factor: a useful tool for the histochemical analysis of specific binding sites.
AuthorsKayser K, Weisse G, Gabius HJ, Hintze T
JournalHistochem J
PubMed ID2228731
Epidermal growth factor (EGF) was labelled with biotin via modification of either the amino or carboxyl groups, using suitable reagents, namely biotinyl-N-hydroxysuccinimide ester or biotinamidocaproyl hydrazide. To assure that the specific binding capacity of EGF is retained despite its chemical modification, displacement of the EGF by biotinylated derivatives in a ... More