Development of a Ligand Blot Assay Using Biotinylated Live Cells

Author:

de Almeida Dias Felipe1,Souza dos Santos André Luis1,Ferreira-Pereira Antônio1,Romeiro Alexandre2,Teixeira Zimmermann Luciana3,Cunha do Nascimento Michelle Tanny1,Correa Atella Georgia4,Saraiva Elvira Maria1,Linden Rafael2,Hampshire Lopes Angela5

Affiliation:

1. Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

2. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

3. Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

4. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

5. Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,

Abstract

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid—sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions. ( Journal of Biomolecular Screening 2007:1006-1010)

Publisher

Elsevier BV

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