Optical properties of Alexa 488 and Cy5 immobilized on a glass surface.
AuthorsWang L, Gaigalas AK, Reipa V
JournalBiotechniques
PubMed ID15679095
'The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret ... More
Multiple site-specific in vitro labeling of single-chain antibody.
AuthorsRamakrishnan B, Boeggeman E, Manzoni M, Zhu Z, Loomis K, Puri A, Dimitrov DS, Qasba PK,
JournalBioconjug Chem
PubMed ID19507852
'For multiple site-specific conjugations of bioactive molecules to a single-chain antibody (scFv) molecule, we have constructed a human anti HER2 receptor, scFv, with a C-terminal fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. The C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the ... More
A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.
AuthorsBrustad EM, Lemke EA, Schultz PG, Deniz AA,
JournalJ Am Chem Soc
PubMed ID19108697
'A general strategy for the site-specific dual-labeling of proteins for single-molecule fluorescence resonance energy transfer is presented. A genetically encoded unnatural ketone amino acid was labeled with a hydroxylamine-containing fluorophore with high yield (>95%) and specificity. This methodology was used to construct dual-labeled T4 lysozyme variants, allowing the study of ... More
Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.
AuthorsWu P, Shui W, Carlson BL, Hu N, Rabuka D, Lee J, Bertozzi CR,
JournalProc Natl Acad Sci U S A
PubMed ID19202059
'The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the ... More
Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence.
AuthorsFundador E, Rusling J
JournalAnal Bioanal Chem
PubMed ID17206410
'Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) ... More
Use of fluorescence sensors to determine that 2-deoxyribonolactone is the major alkali-labile deoxyribose lesion produced in oxidatively damaged DNA.
AuthorsXue L, Greenberg MM
JournalAngew Chem Int Ed Engl
PubMed ID17154191
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
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
Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling.
AuthorsDirksen A, Dawson PE,
JournalBioconjug Chem
PubMed ID19053314
A high-yielding and rapid chemoselective ligation approach is presented that uses aniline catalysis to activate aromatic aldehydes toward two amine nucleophiles, namely, 6-hydrazinopyridyl and aminooxyacetyl groups. The rates of these ligations are resolved for model reactions with unprotected peptides. The resulting hydrazone and oxime conjugates are attained under ambient conditions ... More
A flexible method for the conjugation of aminooxy ligands to preformed complexes of nucleic acids and lipids.
AuthorsHecker JG, Berger GO, Scarfo KA, Zou S, Nantz MH,
JournalChemMedChem
PubMed ID18666266
Attachment of targeted ligands to nonviral DNA or RNA delivery systems is a promising strategy that seeks to overcome the poor target selectivity generally observed in systemic delivery applications. Several methods have been developed for the conjugation of ligands to lipids or polymers, however, direct conjugation of ligands onto lipid- ... More
Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine.
AuthorsPoon HF, Abdullah L, Reed J, Doore SM, Laird C, Mathura V, Mullan M, Crawford F,
JournalBiol Proced Online
PubMed ID18385803
Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication ... 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