Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.
AuthorsBuck E, Li J, Chen Y, Weng G, Scarlata S, Iyengar R
JournalScience
PubMed ID10037604
'Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within ... More
Incorporation of CpG oligonucleotide ligand into protein-loaded particle vaccines promotes antigen-specific CD8 T-cell immunity.
AuthorsStandley SM, Mende I, Goh SL, Kwon YJ, Beaudette TT, Engleman EG, Fréchet JM
JournalBioconjug Chem
PubMed ID17226959
'The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as ... More
High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast.
AuthorsAjo-Franklin CM, Kam L, Boxer SG
JournalProc Natl Acad Sci U S A
PubMed ID11717428
'Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle ... More
Recovery of sodium dodecyl sulfate-proteins from gel electrophoretic bands in a single electroelution step for mass spectrometric analysis.
AuthorsYefimov S, Sjomeling C, Yergey AL, Li T, Chrambach A
JournalAnal Biochem
PubMed ID10964411
'Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled ... More
CD4+ T cells derived from B cell-deficient mice inhibit the establishment of peripheral B cell pools.
AuthorsBaumgarth N, Jager GC, Herman OC, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID10781082
'We demonstrate that adoptive transfer of peritoneal cavity B cells fails to replenish the peripheral B-1 cells in adult B cell-deficient (mu(-/-)) mice but does replenish adult RAG-1(-/-) mice. We show that this lack of self-replenishment in mu(-/-) mice is mediated by strongly inhibitory, radiation-sensitive CD4(+) T cells that also ... More
11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.
AuthorsDe Rosa SC, Herzenberg LA, Herzenberg LA, Roederer M
JournalNat Med
PubMed ID11175858
Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.
AuthorsBrelje TC, Wessendorf MW, Sorenson RL
JournalMethods Cell Biol
PubMed ID8246789
Cell-free N-terminal protein labeling using initiator suppressor tRNA.
A highly efficient method for the introduction of fluorophores and other markers at the N terminus of proteins produced in a cell-free extract has been developed. The method utilizes an amber (CUA) initiator suppressor tRNA chemically aminoacylated with a fluorophore-amino acid conjugate which is introduced into an Escherichia coli S30 ... More
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
Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins.
Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements ... More
An affinity-based method for the purification of fluorescently-labeled biomolecules.
AuthorsNguyen T, Joshi NS, Francis MB
JournalBioconjug Chem
PubMed ID16848391
Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the ... More
Transfer of SDS-proteins from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel.
AuthorsYefimov S, Yergey AL, Chrambac A
JournalJ Biochem Biophys Methods
PubMed ID10647815
Five SDS-proteins, ranging in molecular weight from 14 to 66 kDa, were detected without covalent fluorescent labeling by the automated gel electrophoresis apparatus with intermittent fluorescence scanning (HPGE apparatus, LabIntelligence) during electrophoresis in barbiturate buffer in the presence of Cascade Blue. The SDS-proteins were electroeluted from the gel into 220 ... More
Fiber-optic immunosensor for measurement of myoglobin.
AuthorsHanbury CM, Miller WG, Harris RB
JournalClin Chem
PubMed ID9365398
A self-contained fiber-optic immunosensor was developed to measure the 16,500-Da protein myoglobin. The sensing element was constructed by entrapment of Cascade Blue-labeled antibody within polyacrylamide gel at the distal face of an optical fiber 300 microns in core diameter. The polyacrylamide gel composition was optimized to allow diffusion of myoglobin ... More
Pairs of violet-light-excited fluorochromes for flow cytometric analysis.
AuthorsAnderson MT, Baumgarth N, Haugland RP, Gerstein RM, Tjioe T, Herzenberg LA, Herzenberg LA
JournalCytometry
PubMed ID9845438
We describe pairs of fluorochromes for use with the 407-nm line of a violet-light-enhanced krypton ion laser. These fluorochromes and a previously described violet-light-excited reporter variant, GFP-Vex, fall into two emission classes: blue for Cascade Blue, and green/yellow for Cascade Yellow, Lucifer Yellow, and GFP-Vex. Cascade Yellow is a new ... More
8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity.
AuthorsRoederer M, De Rosa S, Gerstein R, Anderson M, Bigos M, Stovel R, Nozaki T, Parks D, Herzenberg L, Herzenberg L
JournalCytometry
PubMed ID9415416
We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and ... More
B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection.
AuthorsBaumgarth N, Herman OC, Jager GC, Brown LE, Herzenberg LA, Chen J
JournalJ Exp Med
PubMed ID10899913
We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell-derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus ... More
Identification of a germ-line pro-B cell subset that distinguishes the fetal/neonatal from the adult B cell development pathway.
AuthorsLu LS, Tung J, Baumgarth N, Herman O, Gleimer M, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID11867763
Studies presented here show that the expression of CD4, MHC class II (Ia,) and B220 cleanly resolves a major and a minor subset within the earliest pro-B cell population (germ-line pro-B) in adult bone marrow (BM). The major subset expresses intermediate B220 and low CD4 levels. The minor subset, which ... More
Nine color eleven parameter immunophenotyping using three laser flow cytometry.
AuthorsBigos M, Baumgarth N, Jager GC, Herman OC, Nozaki T, Stovel RT, Parks DR, Herzenberg LA
JournalCytometry
PubMed ID10331625
BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in ... More
Ca2+ regulation of interactions between endoplasmic reticulum chaperones.
AuthorsCorbett EF, Oikawa K, Francois P, Tessier DC, Kay C, Bergeron JJ, Thomas DY, Krause KH, Michalak M
JournalJ Biol Chem
PubMed ID10037706
Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure ... More
Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.
AuthorsWhitaker JE, Haugland RP, Moore PL, Hewitt PC, Reese M, Haugland RP
JournalAnal Biochem
PubMed ID1724118
Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and ... More
Vitronectin interaction with glycosaminoglycans. Kinetics, structural determinants, and role in binding to endothelial cells.
Vitronectin (VN) is a high affinity heparin-binding protein. The physiological role of this binding has hitherto received little attention, and its molecular determinants are subject to controversy. In this study, we characterized vitronectin interaction with heparin, heparin analogues, bacterial extracts, and cell surface glycosaminoglycans. As assessed by (i) fluorescence assays, ... More
Lymphocyte surface thiol levels.
AuthorsSahaf B, Heydari K, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID12642656
Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by ... More
Role of dynamic interactions in effective signal transfer for Gbeta stimulation of phospholipase C-beta 2.
AuthorsBuck E, Schatz P, Scarlata S, Iyengar R
JournalJ Biol Chem
PubMed ID12388553
Heterotrimeric G protein subunits regulate their effectors by protein-protein interactions. The regions involved in these direct interactions have either signal transfer or general binding functions (Buck, E., Li, J., Chen, Y., Weng, G., Scarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). Although key determinants of signal transfer regions for ... More