Reagents and Proteins
1. Succinimidyl-6- biotinamido hexanoate NHS-LC-Biotin Pierce, Rockford, IL . 2. 2- 4'-hydroxyazobenzene benzoic acid HABA Pierce . 3. O-Phenylenediamine OPD, in tablets of 60 mg Sigma, St. Louis, MO . 4. Na51CrO4 Perkin Elmer, Boston, MA . 8. Catalase from bovine liver Calbiochem, CA . 9. Streptavidin from Streptomyces avidinii Calbiochem . 11. Horseradish peroxidase HRP . 12. Monoclonal anti-PECAM antibody clone 62 was generously provided by Dr. Nakada Centocor, Malvern, PA . 13. Mouse IgG...
Buffers Media and Cells
1. Phosphate-buffered saline PBS 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2. 2. HABA stock solution 10 mM HABA in 10 mM NaOH. Add 24.2 mg to 10 mL of 10 mM NaOH. The solution may be stored at 4 C. 3. HABA avidin working solution dissolve 10 mg of avidin in 19.4 mL PBS and add 600 L of 10 mM HABA stock solution. 4. Cell culture medium M199 medium Gibco, Grand Island, NY , 10 fetal calf serum Gibco supplemented with 100 g mL heparin Sigma , 2 mM L-glutamine Gibco , 15 g mL endothelial...
Silvia Muro Vladimir R Muzykantov and JuanCarlos Murciano
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 of targeting. This chapter describes methodologies for evaluating the binding and internalization of labeled conjugates directed to endothelial surface adhesion molecules in cell cultures using anti-intercellular adhesion...
Biotinylation
1. It is important to remember that biotinylation depends on initial concentration of protein. The level of biotinylation is increased at a higher concentration of protein even at same NHS-LC-biotin protein molar ratio. 2. Biotinylation efficiency and its effects on protein activities vary significantly from protein to protein. In case of catalase, biotinylation does not change the catalytic activity at up to a level of 4-5 biotin catalase monomer. Fig. 5. Cell protection against H2O2-induced...
Biotinylation of IgG Antibodies and Catalase
1. Dissolve IgG anti-PECAM antibody or any other mouse IgG and catalase in 0.1 M PBS to concentrations of 3.5 and 5.0 mg mL, respectively. Considering that molecular masses of IgG and catalase are 150 and 240 kDa, respectively, their molar concentrations are 23.3 M and 20.8 M, respectively see Notes 1 and 2 . 2. Prepare fresh 0.1 M NHS-LC-biotin in DMF, that is, dissolve 4.5 mg NHS-LC-biotin in 100 L of anhydrous DMF. Keep solutions of proteins and NHS-LC-biotin on ice. 3. Add appropriate...
M E T H O D S I N M O L E C U L A R B I O L O G Y 1
Fachbereich Chemie der Universit t Dortmund Dortmund, Germany 2004 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology is a trademark of The Humana Press Inc. All papers, comments, opinions,...
Results
Catalase was biotinylated to a level of 3.25 biotin catalase monomer or approx 12-13 biotin catalase tetramer and monoclonal anti-PECAM antibody was biotinylated to 3.5 biotin IgG. Both proteins were mixed at a molar ratio 1 1, and streptavidin was added to form conjugates. The titration curve of the conjugation is shown in Fig. 2A as a dependence of conjugate effective diameter determined by DLS on molar ratio streptavidin biotinylated proteins. The curve demonstrates a continuous increase of...
M E T H O D S I N M O L E C U L A R B I O L O G Y
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