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Conjugation of Polymers with Biomolecules and Polymeric Vaccine Development Technologies
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
N, N-Carbonyldiimidazole is another direct cross-linking reagent which carries two acylimidazole groups that are separated during conjugation in structure properties (Hermanson 1996; Hermanson 2013).
The Chemical Cross-Linking Of Peptide Chains
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Care must be taken in the size analysis of intramolecularly linked species in any denaturing medium (i.e., sodium dodecylsulfate, guanidine hydrochloride, etc.) since, unless the cross-linking reagent is cleaved by reduction, the intramolecularly cross-linked protein will not denature properly and will probably give falsely low molecular weight results.19,20
Targeted Cytotoxicity
Published in Siegfried Matzku, Rolf A. Stahel, Antibodies in Diagnosis and Therapy, 2019
Uwe Zangemeister-Wittke, Winfried Wels
The antibody is usually modified with a heterobifunctional cross-linking reagent to introduce a selectively reactive group useful for conjugation. Most often an alkylating function or a sulfhydryl group have served this purpose. Dependent on its structure, the toxin may also be modified. In proteins, several amino acid side chains contain reactive groups that can serve as sites of attachment for cross-linking reagents (described in detail by Wawrzynczak and Thorpe, 1987). The ε-amino group of lysine, which is present on the surface of most proteins, is especially suitable for cross-linking since it reacts with a number of different reagents under conditions that do not affect other chemical groups in the protein. 2-iminothiolane (2-IT) has been frequently used to introduce sulfhydryl groups into primary amines for the reaction with free thiols or maleimide residues provided by cross-linking reagents, to form reducible disulfide bonds or stable thioether bonds, respectively. The native thiol groups of cysteine residues represent other suitable target sites for cross-linkers containing alkylating groups. Alternatively, the cysteinyl thiol group can be reacted with thiol-containing cross-linkers to form disulfide bonds.
Synthesis and characterisation of aqueous haemoglobin-based microcapsules coated by genipin-cross-linked albumin
Published in Journal of Microencapsulation, 2020
Kai Melvin Schakowski, Jürgen Linders, Katja Bettina Ferenz, Michael Kirsch
The determination of Hb concentrations in the stock solution, the first supernatant and the supernatants after each washing step showed that there was hardly any Hb traceable in the supernatants after each washing step. This result implied that Hb is tightly enclosed by MnCO3 and that possible pores inside of MnCO3 particles must be chiefly smaller than the diameter of Hb proteins. The resulting encapsulation efficiency of Hb inside MnCO3 particles varied from 39% to 82% depending on the amount of Hb initially added to the Na2CO3 and MnCl2 solutions. The size of the resulting particles did not differ in dependence on the amount of cross-linking reagent added before dissolution of the MnCO3 core. For genipin as cross-linking reagent concentrations of 1, 2, 3 and 4 mM were chosen, all leading to capsules measuring 3–4 μm in diameter as measured by DLS (s. Table 1).
Ecto-F0/F1 ATPase as a novel candidate of prothymosin α receptor
Published in Expert Opinion on Biological Therapy, 2018
Hiroshi Ueda, Hayato Matsunaga, Yosuke Matsushita, Shiori Maeda, Ryusei Iwamoto, Shigeyuki Yokoyama, Mikako Shirouzu
In our previous attempts to identify GPCR-type ProTα binding proteins by use of affinity-cross-linkage to brain membranes, we have failed to detect any GPCR-like protein bands from brain membranes. In the present study, we chose the Gαi-rich lipid rafts preparation as the source of ProTα-target proteins of N18-RE105 cells. Figure 1(A) showed that Gαi-like immunoreactivities were abundantly found in lanes 6–11. On the analogy of previous report on Thβ4 protein, we performed the western blotting experiments of streptavidin sepharose-purified biotinylated ProTα-binding proteins by use of anti-ATP5A1 or anti-ATP5B antibody. For this purpose we used Sulfo-SBED, a trifunctional cross-linking reagent having biotin covalently attached to a heterobifunctional reagent. In this reaction, the first step was the replacement of sulfonated N-hydroxysuccinimide moiety with ProTα followed by the photo-affinity cross-link with target binding proteins for ProTα. After the reduction of reacted products, biotinylated target binding proteins were separated by SDS-PAGE. As seen in Figure 1(B), significant immunoreactive signals by anti-ATP5B antibody were observed at 58 kDa in lanes 13–16, while any significant signals by anti-ATP5A1 antibody were not observed at ~55 kDa in these lanes. However, the possibility that ProTα may bind to other target proteins cannot be excluded, since no attempt to detect immunoreactive biotinylated proteins has been done. Pooled samples (fractions 1–5, 6–11, and 12–16), which have been pulled-down by use of streptavidin sepharose were separated on SDS-PAGE and silver-stained. A major band at ~55 kDa was digested by trypsin and analyzed by MALDI-TOF-MS/MS. From the data of 14 digested peptides (Figure 1(D)), it was identified to be F1-ATPase β-subunit ATP5B.