Optimizing antibody immobilization strategies for the construction of protein microarrays.
AuthorsPeluso P, Wilson DS, Do D, Tran H, Venkatasubbaiah M, Quincy D, Heidecker B, Poindexter K, Tolani N, Phelan M, Witte K, Jung LS, Wagner P, Nock S
JournalAnal Biochem
PubMed ID12531195
'Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function ... More
N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts.
AuthorsAtamna H, Paler-Martínez A, Ames BN
JournalJ Biol Chem
PubMed ID10702229
'Alpha-phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice. N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PBN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 ... More
Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay.
AuthorsKow YW, Dare A
JournalMethods
PubMed ID11020331
'Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites. Many procedures are available for the quantification and detection of base damage and AP sites. However, either these procedures are laborious or the starting materials are difficult to obtain. A biotinylated aldehyde-specific ... More
Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy.
AuthorsHirose T, Ohtani T, Muramatsu H, Tanaka A
JournalPhotochem Photobiol
PubMed ID12194206
'A new method was developed to allow direct visualization of damaged sites on individual DNA molecules. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labeled with a biotinylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. The light emitted by a ... More
A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications.
AuthorsKhidekel N, Arndt S, Lamarre-Vincent N, Lippert A, Poulin-Kerstien KG, Ramakrishnan B, Qasba PK, Hsieh-Wilson LC
JournalJ Am Chem Soc
PubMed ID14692737
Genetic introduction of a diketone-containing amino acid into proteins.
AuthorsZeng H, Xie J, Schultz PG
JournalBioorg Med Chem Lett
PubMed ID16934461
An orthogonal tRNA/aminoacyl-tRNA synthetase pair was evolved that makes possible the site-specific incorporation of an unnatural amino acid bearing a beta-diketone side chain into proteins in Escherichia coli with high translational efficiency and fidelity. Proteins containing this unnatural amino acid can be efficiently and selectively modified with hydroxylamine derivatives of ... More
High-efficiency labeling of sialylated glycoproteins on living cells.
AuthorsZeng Y, Ramya TN, Dirksen A, Dawson PE, Paulson JC,
JournalNat Methods
PubMed ID19234450
We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of ... More
Repair kinetics of abasic sites in mammalian cells selectively monitored by the aldehyde reactive probe (ARP).
AuthorsAsaeda A, Ide H, Tano K, Takamori Y, Kubo K
JournalNucleosides Nucleotides
PubMed ID9708359
Human methylpurine N-glycosylase (MPG) activity was investigated by monitoring abasic (AP) sites resulting from removal of alkylated bases. The amount of AP sites in MMS-treated HeLa cells transiently increased at 3 h, then gradually decreased to 40% at 24 h. The presence of adenine, an inhibitor of AP endonucleases, in ... More
Regioselective labeling of antibodies through N-terminal transamination.
AuthorsScheck RA, Francis MB
JournalACS Chem Biol
PubMed ID17455901
A convenient new method is described for the introduction of ketone groups at the N-termini of antibodies. The reaction occurs in the presence of pyridoxal-5'-phosphate under conditions mild enough to maintain antigen binding function, as confirmed by enzyme-linked immunosorbent assay. Further derivatization of these functional sites was accomplished through oxime ... More
Detection of abasic sites on individual DNA molecules using atomic force microscopy.
AuthorsSun HB, Qian L, Yokota H
JournalAnal Chem
PubMed ID11393845
We have developed an atomic force microscopy-based method for detecting abasic sites (AP sites) on individual DNA molecules. By using uracil and uracil DNA glycosylase, we first prepared a 250-bp DNA template consisting of two AP sites at specific locations. We then detected the AP sites by marking them with ... More
New role for an old probe: affinity labeling of oxylipid protein conjugates by N'-aminooxymethylcarbonylhydrazino d-biotin.
AuthorsChavez J, Wu J, Han B, Chung WG, Maier CS,
JournalAnal Chem
PubMed ID17007505
Free radicals, electrophiles, and endogenous reactive intermediates are generated during normal physiological processes and are capable of modifying DNA, lipids, and proteins. However, elevated levels of oxidative modifications of proteins by reactive species are implicated in the etiology and pathology of oxidative stress-mediated diseases, neurodegeneration, and aging. A mass spectrometry-based ... More
Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection.
AuthorsBoeggeman E, Ramakrishnan B, Pasek M, Manzoni M, Puri A, Loomis KH, Waybright TJ, Qasba PK,
JournalBioconjug Chem
PubMed ID19425533
The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the ... More
Protein micropatterns using a pH-responsive polymer and light.
AuthorsChristman KL, Maynard HD
JournalLangmuir
PubMed ID16114947
Protein and peptide microarrays are popular candidates for medical diagnostics because of the possibility for high sensitivity and simultaneous marker screening. To realize the potential of these arrays, new strategies for ligand patterning are needed. We report a method for patterning proteins that utilizes a pH-responsive polymer, deep ultraviolet (DUV) ... More
Submicron streptavidin patterns for protein assembly.
AuthorsChristman KL, Requa MV, Enriquez-Rios VD, Ward SC, Bradley KA, Turner KL, Maynard HD
JournalLangmuir
PubMed ID16893251
Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure ... More
A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.
AuthorsKubo K, Ide H, Wallace SS, Kow YW
JournalBiochemistry
PubMed ID1567824
Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA ... More
Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method.
AuthorsBoeggeman E, Ramakrishnan B, Kilgore C, Khidekel N, Hsieh-Wilson LC, Simpson JT, Qasba PK
JournalBioconjug Chem
PubMed ID17370997
The mutant beta1,4-galactosyltransferase (beta4Gal-T1), beta4Gal-T1-Y289L, in contrast to wild-type beta4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc ... More
A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.
AuthorsAtamna H, Cheung I, Ames BN
JournalProc Natl Acad Sci U S A
PubMed ID10639140
Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ... More
Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA.
AuthorsIde H, Akamatsu K, Kimura Y, Michiue K, Makino K, Asaeda A, Takamori Y, Kubo K
JournalBiochemistry
PubMed ID8347625
The abasic site (apurinic/apyrimidinic site) is the most common lesion in DNA and is suggested to be an important intermediate in mutagenesis and carcinogenesis. We have recently reported a novel assay for the detection and quantitation of abasic sites in DNA [Kubo, K., Ide, H., Wallace, S. S., & Kow, ... More