生物化学技术

Epidermal growth factor-like (EGF) repeats are found in numerous extracellular or transmembrane proteins including Notch. EGF repeats containing the appropriate consensus sequences can be modified with two unusual types of glycans: O-fucosylation and O-glucosylation. We ha ...

A fast and economical procedure for the production of peptide nucleic acid (PNA) microarrays is presented. PNA oligomers are synthesized in a fully automatic manner in 96-well plates using standard Fmoc chemistry. Subsequently, the oligomers are released from the support and spotted on ...

This method describes the use of subtilisin-catalyzed peptide condensation to form a 15-residue glycopeptide from two smaller synthetic peptides. A 12-residue peptide ester is synthesized by solid-phase peptide synthesis using a PAM-modified Rink amide resin that allows the form ...

This method describes the conjugation of a synthetic glycopeptide to the N-terminus of a recombinant human interleukin-2 (IL-2) protein fragment. The IL-2 protein fragment is produced as an affinity-tagged fusion protein in Escherichia coli and then cleaved with the highly selective T ...

Chemically synthesized truncated Ras proteins are coupled to C-terminal Ras peptides via a maleimidocaproyl linker. The resulting product is isolated by extraction with Triton X-114. The biological activity of these oncogenic Ras lipoproteins can be determined in a cell-based dif ...

Expressed protein ligation is a powerful tool for the generation of natively folded proteins composed of recombinantly generated and chemically synthesized polypeptides. Using this approach, we developed protocols for the production of prenylated and/or otherwise-labeled ...

This chapter describes the hydrophobic modification of peripheral membrane-anchored proteins by isoprenylation and S-acylation. The coupling of bacterially expressed protein moieties with chemically synthesized lipopeptides is described as an in vitro alternative f ...

Two unusual enzymatic activities are required for the realization of a single molecule sequencing: a polymerase for copying a deoxyribonuclease (DNA) target into complementary flurophore-labeled DNA, and an exonuclease for the successive hydrolysis of the completely dye-lab ...

Three fluorophor-labeling methods for gene expression profiling on deoxyribonucleic acid (DNA) microarrays are described. All three techniques start from total ribonucleic acid (RNA) samples. Two procedures are based on first-strand complementary DNA synthesis by reverse t ...

Synthetic oligonucleotides are versatile tools for recognizing ribonucleic acid and deoxyribonucleic acid. This chapter describes methods for enhancing recognition by derivatizing oligonucleotides with either proteins or peptides.

The conjugation of macromolecules offers a rapid and versatile route to improved function. Here, the methods for obtaining disulfide-linked oligonucleotide-peptide and oligonucleotide-protein conjugates are described. These hybrid molecules can be used deliver chemic ...

Semisynthetic DNA-protein conjugates are synthesized by covalent coupling of thiol-modified DNA oligonucleotides and streptavidin. The resulting conjugates have a binding capacity for four equivalents of biotin and one nucleic acid of complementary sequence. The conjuga ...

The study of biological processes relies increasingly on methods for probing structure and function of biochemical machinery (proteins, nucleic acids, and so on) with submolecular resolution. Atomic force microscopy (AFM) has recently emerged as a promising approach for imaging b ...

Gold nanoparticles functionalized with thiol-modified single-stranded oligonucleotides are highly useful reagents for a variety of applications, ranging from materials science to bioanalytics. In this chapter, the preparation of citrate stabilized 15-nm Au nanopartic ...

The streptavidin-biotin system may be used to synthesize immunoconjugates for targeted delivery of drugs, including therapeutic enzymes. The size of antibody-enzyme conjugates, which is controlled by the extent of biotinylation and molar ratio between the conjugate componen ...

Streptavidin-biotin conjugates of enzymes with carrier antibodies provide a versatile means for targeting selected cellular populations in cell cultures and in vivo. Both specific delivery to cells and proper subcellular addressing of enzyme cargoes are important parameters ...

The conjugation of stimuli-responsive, or “smart,” polymers to streptavidin is described. The polymer is synthesized with a thiol-reactive end-group, which is used to end-graft the polymer to cysteine or lysine side-chains that are genetically engineered into controlled positions ...

Heterobifunctional crosslinking reagents are small molecular weight chemicals containing two different reactive groups that have become important tools in generating conjugates of two different biomolecules, such as two proteins. The resulting bioconjugates are hybrid ...

This chapter discusses two related methods for creating Fab′ � Fab′ chemically linked BsAb. Both methods require the generation of purified F(ab′)2 fragments of each antibody and use reagents that react with the free thiols generated upon reduction of interheavy chain disulfide bonds of the F ...

This chapter provides a critical overview of the technology presently available in the field of protein PEGylation. The chemistry of the polymer and of its reactive derivatives is discussed and presented together with several protocols used to obtain PEG-protein conjugates. The coup ...